BioSoft | Biophysics and Soft-Matter

June 9, 2018 | Author: Jonturk Jonturk | Category: Biophysics, Cell Membrane, Ion Channel, Viscosity, Cytoskeleton


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Mitglied der Helmholtz-GemeinschaftBioSoft Selected results 2007/2008 Contents Editorial Introduction Selected Results Institute of Solid State Research (IFF) Theoretical Soft Matter and Biophysics Neutron Scattering Soft Matter page 3 page 5 page page page page page page page 7 9 27 43 59 73 89 Institute of Structural Biology and Biophysics (ISB) Cellular Biophysics Molecular Biophysics Structural Biochemistry Institute for Bio- and Nanosystems (IBN) Bioelectronics Biomechanics page 105 page 115 page 129 Education & Dissemination Publications page 139 1 Impressum BioSoft – Selected Results 2007/2008 Herausgeber: Forschungszentrum Jülich GmbH Stabsstelle Fachstrategie Institut für Festkörperforschung 52425 Jülich Deutschland +49-2461-61-3026 ¬ +49-2461-61-3570 www.fz-juelich.de Redaktion: Dr. Wolfgang Speier Kurt Wingerath Druck: Grafische Medien Forschungszentrum Jülich GmbH Juni 2009 © Forschungszentrum Jülich GmbH 2 The success of this spring school made clear to us that a more long-term effort is needed to provide an interdisciplinary graduate education. jointly use modern experimental techniques and instrumentation. in order to equip students and young researchers with the necessary techniques and knowledge to address the many exciting scientific challenges at the interface between biology. In parallel. the Institute for Bio. At the Forschungszentrum Jülich.and Nanosystems (IBN) and the Institute of Structural Biology and Biophysics (ISB). We are very happy that Erwin Neher. and work on several joint research projects. which illustrate the research in SoftMatter and Biophysics in the Forschungszentrum Jülich during the last two years. Scientist from these institutes share many common interests. Research in Soft-Matter and Biophysics spans over three institutes on Jülich campus: the Institute of Solid State Research (IFF). physicists and biologists have started to intensify their collaboration in 2004 by organizing a two-week IFF spring school with the title “Physics meets Biology”. was among the lecturers of this school. together with colleagues from the Universities of Düsseldorf and Köln. chemistry and physics. The current brochure presents a selection of reports. we have continued to educate a wider range of students by organizing spring schools. This school was attended by an international audience of about 200 students. the Nobel laureate and inventor of the patch-clamp technique (together with Bert Sakmann). Gerhard Gompper 3 .Editorial “Physics meets Biology” Physicists and Biologists worldwide have recognized some time ago that they can benefit a lot from sharing their knowledge and working together to understand the behavior of complex systems. The research center CAESAR in Bonn has joined the graduate school recently. in particular in 2008 by a school entitled ``Soft Matter — From Synthetic to Biological Materials". This has lead the foundation of the International Helmholtz Research School on Biophysics and Soft Matter (IHRS BioSoft). supported by a grant from the Helmholtz Association. The school accepted its first students in the fall 2006. 4 . mechanical and electrical stimuli. cells constantly receive. in a strictly defined linear sequence. The development and fate of every single cell is regulated on the molecular level by a variety of cross-linked mechanisms. Advanced physical tools and quantitative methods provide new routes in the investigation of biological molecules and systems.most sensitive methods available. The aim of Soft Matter research is to understand the cooperative behaviour of macromolecular systems with many interacting degrees of freedom. nanobiotechnology. Soft Matter encompasses all kinds of macromolecular assemblies of polymeric. e. pathogens or environmental influences. Prominent examples are linear polymers. thermal fluctuations compete with conservative forces arising. or direct structure formation. and thereby taylor material properties. This is especially true for the extremely specific interactions between proteins. for example. and with topological constraints. and life and health sciences. the molecules of life. cross linking. Here. process. building blocks. This implies that such fields can be employed to modulate. are linear chain molecules. which are not present in small molecules. Thermodynamics drives the selfassembly of macromolecular building blocks into mesoscopic structures with a large variety of rheological. assist. Also. Worldwide. Even simple synthetic polymers that are typically made from identical monomers allow the design of complex material properties that depend. This requires a highly multidisciplinary infrastructure combining medium and large-scale experimental facilities with scientific computing and a high-level laboratory science. Biophysics seeks to understand the processes of life by applying advanced physical methods to complex biological systems. and emit information such as chemical. the efficiency of interlinked signaltransduction chains and of information processing is enormously high. A profound. we experience a rapid convergence of physical and biological research areas and possibly also research structures. Knowledge and techniques created by cutting-edge research in this transdisciplinary field is essential for the sustained rapid progress in nanotechnology. On the cellular level. Biological components or biological construction principles can be used or mimicked in the design of new materials. and systems level. which are constructed from a single or a few kinds of building blocks. nucleic acids and proteins. electronic. e. on their lengths. This understanding requires exquisitely detailed and quantitative knowledge of the underlying processes at the molecular. Large mesoscopic length scales imply long time scales. structural biology aims at obtaining precise geometric and dynamic information about biologically and medically relevant molecules as a basis for an understanding of their molecular functions. nonequilibrium behavior is ubiquitous in soft matter systems driven by external fields or hydrodynamic flows. There are several compelling reasons for this approach: ƒ ƒ ƒ ƒ A profound understanding of fundamental mechanisms underlying biological function and self-organisation can be fostered by studies of simpler synthetic soft matter systems. quantitative understanding of selected model systems ranging from molecules to cells lays the foundations for modern systems biology. Therefore.g. respectively. thermal. Therefore. cellular. Soft matter physics and biophysics represent a very dynamic and rapidly growing area of multidisciplinary research at the interface between physics. spontaneous mutations. colloidal.. magnetic. Thus. because malfunctions of cellular information processing caused by endogenous or exogenous factors.. 5 .g. Due to the weak interactions between building blocks. from van der Waals or Coulomb interactions. and slow dynamical behavior. a detailed understanding is very important. Biophysics and soft matter physics are concerned with the qualitative and quantitative description of structure and dynamics of complex macromolecules and their assemblies at various levels up to living cells.Introduction BioSoft – Biophysics & Soft Matter Macromolecules consisting of hundreds to thousands of atoms have many emergent properties. it is obvious that biological macromolecules have much more complex properties. materials sciences. Highly advanced methods often requiring large-scale facilities are used to investigate their fascinating properties. and potential combination with other polymers. or amphiphilic character. By interaction with their environment. the investigation of which requires the most advanced and – due to production limits . or mechanical properties. consisting of four and twenty different building blocks. Many interesting properties of macromolecules are based on their complex interactions among each other. especially soft nanoscience. photonic. chemistry and biology. and to provide the scientific basis for a rational design of functional nano-scale materials. can lead to serious diseases or to accelerated ageing processes. 6 . and Nanosystems (IBN) Bioelectronics Biomechanics 2007 2008 7 .Selected Results Institute of Solid State Research (IFF) Theoretical Soft Matter and Biophysics Neutron Scattering Soft Matter Institute for Structural Biology and Biophysics (ISB) Cellular Biophysics Molecular Biophysics Structural Biochemistry Institute of Bio. 8 . 9 . both in equilibrium and under flow conditions. which is then linked to the molecular architecture. and exact solutions are employed. A characteristic feature of soft-matter research is the fruitful interaction between theory and experiment. mesoscale hydrodynamic simulation techniques. simulation methods (Monte Carlo. a combination of analytical and numerical methods is often required to successfully characterize the properties of these complex systems. a large variety of methods are applied. Gerhard Gompper The main research topic of the Institute “Theoretical Soft Matter and Biophysics“ is the theory of macromolecular systems. Richter) and the Institute for Soft Condensed Matter (Prof. Soft matter physics and biophysics are interdisciplinary research areas encompassing statistical physics. but also composite systems ranging from colloids in polymer solutions to mixtures of surfactants and amphiphilic block copolymers. colloidal suspensions. In fact. IFF-2 closely cooperates with the Institute for Neutron Scattering (Prof. field theory. molecular dynamics). Dhont) to successfully tackle many of the essential aspects of the systems investigated. vesicles and cells. materials science.IFF-2: Theoretical Soft-Matter and Biophysics Director: Prof. and biology. In particular. Since the building blocks of soft matter systems often contain a large number of molecules. membranes. “simplified“ mesoscale modelling is typically required. chemistry. Our systems of interest include polymer solutions and melts. A major focus is the hydrodynamic behaviour of complex fluids and biological systems. perturbation theory. At IFF-2. and does not conserve angular momentum. or alternatively. Subsequently. Here. i. lattice methods generally suffer from the lack of Galilean invariance. which is often called stochasticrotation dynamics. such as multi-phase flows in Couette geometry. ∂xβ ∂xα ∂xβ ∂xα where α. e. one is faced with the challenge of bridging the gap between the mesoscopic length and time scales of the solute and the atomic scales of the solvent. the last term vanishes (i. y. In the streaming step. membranes. Götze . When a fluid is incompressible. In order to clarify the effects of angular-momentum conservation [2]. We focus on particlebased simulations. Here. multi-particle collision dynamics (MPC) [1]. where an angularmomentum conserving variant is available. The algorithm is constructed in such way that mass. In conventional viscous fluids that do conserve angular momentum. and chemical reaction systems. Thus. G. Noguchi . Thus. Since the equations of continuity and velocity evolution are of the same form. Gompper IFF-2: Theoretical Soft-Matter and Biophysics The angular momentum is conserved in fluids – with a few exceptions such as ferrofluids in external fields. energy and translational momentum are locally conserved. where angular momentum conserving (MPC-AT+a) and nonconserving (MPC-AT−a) algorithms are available [3]. In simulations of the hydrodynamic behavior of complex fluids. MPC has been applied to various systems such as colloids. the particles move ballistically. this is the normal Navier-Stokes equation with viscosity η = η ¯+η ˇ. However. λ is the second viscosity coefficient. ternary amphiphilic fluids. needs less computational time compared to other particle based methods such as DPD. Moreover. H. a well-established. β ∈ {x. thus allowing simulations of larger systems. This symmetry is required by the fact that there is no stress expected in a uniformly rotating fluid (rigid body rotation). it can be violated locally in fluid simulations to reduce computational costs. O. they are sorted into collision cells and the collision step then mimics the simultaneous interaction of all particles within each cell by assigning them new velocities. a coarse-grained mesoscopic fluid model is required that is sufficiently simple to be tractable but still captures the correct hydrodynamic behavior. η ˇ = 0) in angularmomentum-conserving systems. the number of degrees of freedoms can be reduced considerably. We show that there are situations of practical relevance. polymers. by the conservation of angular momentum. we mainly use the Andersen-thermostat version of MPC. the fluid is represented by point particles that undergo subsequent streaming and collision steps.Relevance of Angular Momentum Conservation in Fluid Simulations I. the viscous stress is given by σαβ = + λ(∇ · v)δαβ (1) „ « „ « ∂vα ∂vβ ∂vα ∂vβ η ¯ + +η ˇ − . On the other hand. The effects of this violation are investigated using multi-particle collision dynamics. where angular momentum conservation is essential to avoid non-physical results. the above argument is no longer valid and we have to consider in general an asymmetric tensor. σαβ = σβα . a full treatment on a microscopic level is prohibited by the enormous number of involved particles and the large necessary time range. highly efficient hydrodynamics simulation technique. and η ¯ and η ˇ are the symmetric and asymmetric components of the viscosity. where particle positions and velocities are continuous variables that are updated at discrete times. respectively. the negligence 10 . while the microscopic details of the solvent that mediates the hydrodynamic interactions are rather unimportant. which can be categorized in lattice methods or particle-based methods. the angular momentum is not conserved in the most widespread version of MPC. Moreover. for a fluid without conservation of angular momentum. which is essential for correct hydrodynamic behavior. Hybrid simulations combining an MPC fluid with molecular dynamics of solute particles are easily possible. However. (2) Dt where D/Dt is Lagrange’s derivative and P is the pressure. coupling to solute particles as well as moving boundaries can be treated more easily than in lattice methods and thermal fluctuations are naturally contained. The equation of velocity evolution is given by ρ Dv = −∇P +(λ + η ¯− η ˇ)∇(∇· v)+(¯ η +η ˇ)∇2 v. e. By representing a large number of physical solvent molecules by one model fluid particle at a time. (1) is linear in the vorticity ∇ × v. Various mesoscopic approaches have been proposed in the last decades. the viscous stress tensor has to be symmetric. often only the dynamics of the colloidal particles are of particular interest. As these typically differ by orders of magnitude. The method used here. Then. z }. Here. The last term in Eq. when the boundary conditions on walls are given by forces. We consider a fluid confined between two coaxial cylinders (Couette flow) rotating with the same angular frequency Ω. it generates an additional torque.of angular-momentum conservation does not modify the velocity field of fluids when the boundary conditions are given by velocities. The finite size of the collision cells leads to a correction term. 2: Azimuthal velocity vθ of binary fluids in a rotating cylinder. FIG. This is caused by the asymmetric stress term 2ˇ η Ω. 2). The theoretical velocity profile is obtained from the stress balance at r = R1 and is in very good agreement with the numerical results (Fig. we study fluids confined between eccentric cylinders with fixed axes. and G. in simulations without angularmomentum conservation. in MPC-AT−a fluids. It is assumed that the cylinder rotates very slowly. [2] I. N. 1. (4) 4R In order to verify that the velocity field is not affected by the lack of angular-momentum conservation if the boundary condition is given by the velocity.out (R) = ±4π η ˇΩR2 1 ∓ . As expected. as this corresponds to the rotation of a rigid body. (3). 046705 (2007). We choose the fluid inside (r < R1 = 5a) to have a higher viscosity than the fluid outside. 78. Solid lines represent the analytical results for MPC-AT−a. [3] H. which is impenetrable to the fluid particles. Gompper. 8605 (1999). θ. induced by the stress term of the asymmetric viscosity η ˇ in Eq. The MPC collision performed in cells crossing the boundary propagates the momentum from one fluid to the other. Phys. 1: Stream lines for fluid confined between eccentric cylinders (inner one rotating with constant angular velocity). Gompper. Noguchi. and G. which are shown in Fig. Europhys. and the outer cylinder with radius R2 rotates with constant velocity Ω0 . Kapral. we consider binary fluids with a fixed geometry of the boundary surface. no torque is expected to be acting on the cylinders in angular-momentum conserving fluids. Rev. O. where the inner one is rotating at constant angular velocity. the inner and outer stresses do not coincide. 110. fluids with different viscosities are in contact. the corresponding stream lines. so that the velocity field can be changed when the boundary condition is given by forces. z ). The inner cylinder of radius R1 of circular Couette flow is replaced by a more viscous fluid. In MPC-AT+a. Götze. the azimuthal stress is given by σrθ = (¯ η+η ˇ) r∂ (vθ /r) vθ + 2ˇ η . 2). Chem. in MPC-AT−a. However. Malevanets and R. The occurrence of a back-flow in the large-gap region as well as the resulting total forces on the inner cylinder are in good agreement with theoretical predictions. ∂r r (3) The first term is the stress of the angular-momentumconserving fluid. or finitesized objects rotate in fluids. A boundary of a fluid does not only exist on solid objects but also between two fluids or on membranes. Symbols represent the simulation results of MPC-AT−a for two different viscosity ratios (+. [1] A. However. In order to investigate the fluid-fluid boundary 11 . If both fluids rotate at the same angular velocity. H. However. or two liquids separated by a membrane. The torque is the tangential stress 2ˇ η Ω0 multiplied by the circumference length 2πR and the radius R. ×). FIG. In cylindrical coordinates (r. respectively. and that the flow stress does not change the shape of the interface. Our main conclusion is that simulations which do not conserve angular momentum can lead to quantitatively and even qualitatively incorrect results. Noguchi. J. E 76. which depends on the derivative of the angular velocity Ω = vθ /r. 10005 (2007). the inner fluid rotates more slowly (see Fig. Lett. This is a simplified description of oil and water phase-separated due to surface tension. are practically identical for both methods. both fluids rotate with Ω0 independent of their viscosities. Kikuchi. and MPC-AT+a (◦). The second term is the additional stress from the negligence of angularmomentum conservation and is proportional to Ω. The fluids with different viscosities are located at r < 5a and 5a < r < 10a. Phys. so that the torques on the confining cylinders are found to be „ « 3a Tin. Here. we do observe torques on the confining cylinders. respectively.g. and η the viscosity. Recent FCS studies raised the question whether dsDNA dynamics in dilute solution is controlled by hydrodynamic interactions [4] or not [5]. dsDNA fragments exhibit rodlike. and more advanced models. E. [4]. In the absence of additional photophysical processes and chemical reactions. semiflexible. the classical experimental techniques. Moreover. the diffusional motion of segments as well as that of the overall molecule can be studied at nanomolar concentration under (quasi)equilibrium conditions in solution and cellular systems. Ohrt 2 . simple generic models of polymer dynamics [1] are not expected to provide a quantitative description of dsDNA behavior. P. With ˙ ¸ the mean square displacement (MSD) ∆r2 (t) of a particle. P.. the FCS correlation function assumes the form 1 1 G (t) = . T. accounting for the persistence of the polymer chain. predominantly deliver information on large-scale shape fluctuations of macromolecules. kB the Boltzmann factor. depending on their length. where T is the temperature. Our FCS measurements of lengths L from 102 to 2×104 base pairs (bp) (L/lp ∼ 0. and length. We employed the technique of polymerase chain reaction (PCR) to produce monodisperse samples of DNA fragments with predefined sequence. Schwille 2 1 2 IFF-2: Theoretical Soft-Matter and Biophysics Institute of Biophysics/BIOTEC. are required. Dresden University of Technology. Petrov 2 . the semiflexible polymer model of Ref. ψn (L/2) 12 . analytical calculations yield the MSD ∞ “ ” ˙ 2 ¸ 2kB T X 2 ˜n ∆r (t) = 6Dt+ τ n ψn (L/2) 1 − e−t/τ πη n=1 (2) for the chain end [4]. where r0 and z0 are the lateral and axial extensions of the detection volume. where diffusion and intramolecular dynamics of dsDNA had simultaneously been investigated over the transition range from stiff to semiflexible chains. Fluorescence correlation spectroscopy (FCS) [2] is a single-molecule technique that can provide more detailed information on the macromolecular dynamics than the classical ensemble-based methods. Winkler 1 . Double-stranded (ds) DNA is a biopolymer characterized by a large persistence length lp ∼ 50 nm [3].7 − 140) by following the Brownian motion of the labeled ends of single DNA molecules filled this gap and showed that the experimental data can quantitatively be described by the theory for semiflexible polymer dynamics [3]. Dresden The diffusion and segmental dynamics of doublestranded λ-phage DNA molecules are quantitatively studied over the transition range from stiff to semiflexible chains. fluctuations of the confocally detected fluorescence signal F (t) = F + δF (t) are studied via the correlation function G (τ ) = δF (t) δF (t + τ ) / F 2 . Spectroscopy of fluorescence fluctuations of single-end fluorescently labeled monodisperse DNA fragments unambiguously shows that double-stranded DNA in the length range of 102 − 2 × 104 base pairs behaves as a semiflexible polymer with segmental dynamics controlled by hydrodynamic interactions. Utilizing the Gaussian semiflexible polymer model. (1) „ «r 2 (t) 2 N ∆ r 2 ∆r (t) 1 + 1+ 2 3 3 r2 z2 0 0 where N is the effective number of molecules in the detection volume. The recent progress in molecular biotechnology resulted in a variety of techniques to produce monodisperse DNA fragments. Precise experiments in polymer physics are impossible without well-defined monodisperse polymer samples covering a wide range of molecular weights. As a result. or even flexible polymer behavior. e.Diffusion and Segmental Dynamics of Double-Stranded DNA R. The dynamic behavior of individual macromolecules in solution is governed by chain connectivity and hydrodynamic interactions [1]. However. the fluorescence signal fluctuates as a result of the Brownian motion of fluorescently labeled particles through the detection volume whose shape be approximated by a 3D Gaussian ` can 2 ´ 2 exp −2r2 /r0 − 2z 2 /z0 . which clearly demonstrates that in this length range dsDNA behaves as a semiflexible polymer with strong hydrodynamic interactions. In FCS [2]. Thus. Understanding of polymer dynamics and quantitative verification of polymer theories require detailed information on segmental motion of individual polymer molecules. such as dynamic light scattering (DLS) or transient electric birefringence (TEB). structure. no experimental studies were reported previously. G. fluorescently labeled at the same single end [3]. By fluorescent labelling of individual segments [3] or continuous labelling of the whole molecule [4]. which stimulated the use of DNA as a model compound in studies of polymer dynamics in solution. [5] and leads to a definitive conclusion on the importance of intramolecular hydrodynamic interactions in dsDNA polymer dynamics. τr ∼ L3/2 ) model. Oxford. Krichevsky. The red lines ˙ ¸ indicate the power laws ∆r2 ∼ t and t2/3 . which are introduced in our quantitative analysis of the experimental data [3]. The Zimm regime with ∆r2 ∼ t2/3 . We would like to point out that such an analysis of the shorttime dynamics. 5. Winkler. Gavrinyov. the short-time behavior is given by β = 3/4 [4]. The intramolecular contribution to the MSD is shown by the green lines (bottom). 1986) [2] E. even ˙ for the ¸ longest sample the apparent power law in ∆r2 (t) observed in the range of 100 − 105 µs is to a large extent due to the crossover from intramolecular motion to overall polymer diffusion. S. F. P. whereas at comparable and longer times the intramolecular contribution to the MSD saturates due to the finite size of the polymer coil. Indeed. Rev. dsDNA behaves as a semiflexible polymer with strong hydrodynamic in˙ ¸ teractions . 2: Diffusion coefficients (top) and longest relaxation times (bottom) of dsDNA. Λ = 0. Alon. Phys. only for times shorter than ∼ 103 µs a power law-type behavior can observe in the intramolecular contribution. Lett. FIG. clearly rules out the Rousetype behavior reported for dsDNA in Ref. For semiflexible polymers. 4] with the parameters lp = 50 nm.9. Doi. Petrov. D is the translational diffusion coefficient of the macromolecule. P. direct application of the power laws to the analysis of the MSD time dependencies. respectively. can be misleading. The Theory of Polymer Dynamics (Clarendon Pres. with the exponents β = 1/2 (free draining) and 2/3 (nondraining). 1: Normalized FCS correlation functions (top) and determined mean square displacements (bottom) for λ-DNA fragments of lengths 0. 0. D ∼ L−1/2 . Within the range of lengths studied. State of the Art and Novel Trends in Fuorescence Correlation Spectroscopy (Springer. Figure 2 displays diffusion coefficients and longest relaxation times – the upper part is not readily acces- sible by DLS or TEB – which we obtained by fitting of the normalized FCS curves [3]. P. these power laws are only valid for times much shorter than the longest relaxation time of the polymer. ΛD = 0. Rädler. R. Shusterman. the theoretical expression yields the short-time behavior ˙ ¸ ∆r2 (t) ∼ tβ .6.2. T. is the value of the n-th eigenfunction of a chain of length L at its end. For long and flexible molecules. 0.6. Phys. Petrov.5. 2. Here. J. Berlin. [5].1. Schwille. Rev. τ ˜n is the n-th relaxation time in the presence of hydrodynamic interactions and is related with the free-draining relaxation time τn via τ ˜n = τn /(1 + 3πη ΛHnn ). E 73. Schwille. S. T. 2). [1] M. Keller. Edwards. the diffusion coefficients and relaxation times exhibit approximately the power law behavior D ∼ L−2/3 (note that there is no excluded volume interaction for short DNAs) and τr ∼ L1. our data are in very good agreement with results obtained by other experimental techniques not related to FCS [3] and closely follow the predictions of the semiflexible polymer theory [4]. our results clearly demonstrate that in the range of the lengths studied. 041919 (2006) [5] R. Λ and ΛD are fit parameters. 97. 92. However. Ohrt. and Λ = 0. 1. or ∼ 103 lp (Fig. 258101 (2006) [4] R. still possible in our study for the two longest DNA fragments. G. 1. The red lines are predictions of the semiflexible polymer model [3. At the same time. Rev. 10. P. Thus. O. The blue symbols indicate our FCS data. O. L/lp 1. Lett. The other data (black symbols) are taken from various sources [3]. as shown in Fig. and Hnn is the matrix element of the Rotne-Prager tensor within the preaveraging approximation [4]. 048303 (2004) 13 . and 20 kbp (from left to right). and thus do not follow the predictions of the Rouse (D ∼ 1/L.7 . and τr ∼ L3/2 (or corresponding expressions modified to account for excluded-volume interactions) [1] can be achieved only for long dsDNA molecules with lengths exceeding 105 bp. S. τr ∼ L2 ) nor the Zimm (D ∼ 1/L1/2 .9. and ΛD = 0. Phys. The experimental correlation functions are very well reproduced for the parameters lp = 51 ± 1 nm. 2007) [3] E. G.FIG. respectively [1]. Winkler. Therefore. as has been proposed recently in Ref. in agrement with predictions of the Rouse and Zimm model. which carry out many of the functions essential for their existence. Among the various known biomolecular devices. Although a complete description is not yet available even for the best-characterized system. 2: Structure of the KcsA potassium channel embedded in a water solvated lipid bilayer membrane. M. FIG. We have investigated using computer simulations the molecular mechanism of selectivity and transport of ions through a specific potassium ion channel. a biological nanomachine which acts as a ATP-driven proton pump. Grudinin. Haan IFF-2: Theoretical Soft-Matter and Biophysics Biomolecular machines are large protein complexes whose activities are essential for providing “life” to a biological cell. differentiation. S. Many of these complexes can be described as “molecular machines” or “molecular motors” or “molecular devices”. In many cases the malfunction of these proteins can be a source of disease. This molecular machine is under investigation using elaborate modelling and computer simulations. Nanotechnology is perfectly realized in biological systems. which reside in cell membranes. Living cells are made up of these complexes. are one of the simplest bionanomachines. Cells are essentially biological assemblers that build thousands of custom-designed molecules and construct new assemblers. Only two monomers of the tetrameric protein are shown.-F. not only from an experimental point of view. This view was pioneered by Richard Feynman’s [1] evocative idea of a self-replicating assembler building nanoscale devices atom by atom. depending on their sizes. One of the most fascinating biomolecular machines is the class of vacuolar H+ -ATPases (or V-ATPases).Biological Nanomachines A. Baumgärtner. V-ATPases are multisubunit complexes (Fig. which are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments and proton transport across the cell plasma membrane. Gwan. The V1 and V0 domains are connected by both a central stalk which is thought to play a crucial role in the assumed rotary mechanism of ATP-driven proton transport. considerable progress has been made recently. The V-ATPases are thought to operate by a rotary mechanism in which ATP hydrolysis in V1 drives rotation of a ring of proteolipid subunits in V0 . The theoretical understanding are currently beginning to emerge. The molecular details at atomic resolution are only partly resolved. complexity and tasks [2]. The essential question in understanding biomolecular machines is concerned with the explanation of the macroscopic phenomenology in terms FIG. of the atomic structures and forces involved. and reproduction. 14 . 1: Cartoon representation of the V-ATPase.1) composed of a peripheral domain V1 (yellow and orange) responsible for ATP hydrolysis and an integral domain V0 (blue and grey) responsible for proton translocation. J. ion channels. but also with respect to computational and theoretical achievements. Pfuetzner.A much simpler and much more well characterized bionanomachine is the KcsA potassium channel [3]. we observed subsequent collective cooperative movements of ions. The absence of such an event is explained by the energy barrier imposed by the periodic pore potential on the water molecule. Nanoscience 4. (1) The movements of neighboring ions and water molecules are strongly correlated. The ability of potassium channels to conduct several K+ ions simultaneously in a single file through the narrow pore at levels near the limit of diffusion is usually described in terms of concerted mechanisms.A. (2) Permons are polarized.65 Å and when the oxygen of the water dipol is oriented towards the ion (“polarized bound state”). Theor. Gwan and A. S . Chait and R. our studies [4. The depth of the potential depends on the orientation of the water molecule with respect to the ion. Gulbis. page 282-296. water and carbonyl groups lining the backbone of the pore. In summary. Chem. This implies that a water molecule acts as a “pawl” in a ratchet mechanism. By pulling out the outermost ion from the exit of the pore (Fig. 69 (1998). The figure shows only two monomers of the tetrameric protein complex embedded in a water solvated lipid bilayer membrane.. the high permeation rate at which ion and water molecules pass through the KcsA ion channel is based on the cooperative hopping of pairs of ions and water molecules mediated by the flexible charged carbonyl groups lining the backbone of the channel.2. [4] J. A. These observations provide the basis of an atomistic concept of the molecular mechanism of the multi-ion transport mechanism. by H.Doyle. MacKinnon.3). Baumgaertner. and as the central part of the channel the selectivity filter containing three potassium ions (depicted by balls) and one water molecule between. Rieth and W. ≈ 3 Å. Morais-Cabral. 5] we found [2] A. J. consisting of Coulomb and van der Waals interactions. R. by M. practically matches with the distance of the lowest Coulomb energy UKW (∆z) between one ion and one water molecule. The selectivity filter facilitates the diffusion of potassium ions at rates approaching 108 ions per second under physiological electrochemical gradients. [3] D.L Cohen. one extracellular loop. They mostly moved in pairs or triples (“permons”). This is the movement of a permon. [5] J. 1961. Phys. 15 . F. (3) The movements of permons are rectified. J. M. Sci. three distinct features of the molecular mechanism. in Miniaturisation. 127. One important fact for the understanding of the efficiency of the correlated movements is that the periodicity of the potential in the selectivity filter. One implication is that the transport of ion-water pairs through an ion channel is much more efficient than the transport of ions only. Science 280. New York. the next neigboring ion hops to this vacancy. B. Compt. Am. 50 (2007). Publ. Reinhold. F. 5] have provided useful insights into the structureconductivity relationship. which acts as a valve in the plasma membrane of prokaryotic cells. A. Baumgaertner. in Handbook of Theoretical and Computational Nanotechnology. J. the ion’s neighboring water molecule prefers to maintain its bound state and follows almost simultaneously the preceding ion towards the vacancy. 3: Molecular view of the selectivity filter of the KcsA potassium channel including ions and water molecules. P. T. but the molecular mechanism of it remained elusive. The classical potential energy UKW (∆z) between a single ion and a single water molecule at distance ∆z. It has been suggested that to reach a high conduction rate in a long-pore channel. Feynman. When the outermost ion exits the channel to the periplasmic side. The multi-ion theory has been acepted over decades. [1] R. Gilber. The cutaway view of the atomic structure of the protein complex (in ribbon representation) is shown in Fig. ions must move through the channel pore in a multi-ion fashion : the permeating ions line up in the narrow channel pore and move in a single file through the channel. D. Employing MD simulations on the basis of the Xray structure of the KcsA channel. 2006. Kuo. Gwan and A. Schommers. J. exhibits energy minima at characteristic distances of ∆z ≈ 3 Å (“bound states”). Baumgaertner. A detailed analysis of these movements lead to the development of a simple twodimensional model of ion-water transport. This is known as “the multi-ion permeation process”. and is believed to be a common feature of the ion transportation process in all potassium channels. ed. The structural details of the filter are decisive for ion selectivity and transport. FIG. 045103 (2007). Another interesting observation from our MD simulations is that very rarely a water molecule has been observed to hop back to the vacancy and join the other ion to form a bound state. Each monomer consists of three helices. The absolute minimum of UKW (∆z) ≈ 30 kB T is at distance ∆z = -2. ed. Based on our molecular dynamics simulations [4. Diffusion in a fluid membrane with a flexible cortical cytoskeleton T. Auth 1 , N. S. Gov 2 1 2 IFF-2: Theoretical Soft-Matter and Biophysics The Weizmann Institute of Science, Department of Chemical Physics, P.O. Box 26, Rehovot 76100, Israel The cytoskeleton hinders protein diffusion in the lipid bilayer of the cell’s plasma membrane. We calculate the influence of a flexible network of long-chain protein filaments, which is sparsely anchored to the bilayer, on protein diffusion. We define a potential landscape for the diffusion based on the steric repulsion between the cytosolic part of the protein and the cytoskeletal filaments, using the pressure field that the cytoskeleton exerts on the bilayer. We predict the changes of the diffusion coefficient upon removal of anchor proteins and for a stretched cytoskeleton. Diffusion within the fluid membrane plays an important role for cellular processes, because the cell communicates with its surrounding via its lipid bilayer [1]. For example, diffusion of activated receptor molecules leads to signal amplification and diffusion of adhesion molecules to the contact area is important for cell adhesion. The cytoskeleton, which is in close proximity to the membrane, has been identified to act as a strong regulator of the diffusion within the cellular membrane. For the red blood cell (RBC), several experiments show that the spectrin cytoskeleton slows down translational diffusion. Using single-molecule tracking techniques, small compartments have been found for the diffusion in the cell membrane [2] that may be explained by the cytoskeletal network below the bilayer. Our work focusses on the diffusion in the membrane of cells that have a cortical, two-dimensional cytoskeleton, which is composed of flexible long-chain proteins [3]. A prominent example of such cells are RBCs, where the long-chain proteins are spectrin tetramers. However, a similar cortical cytoskeleton is found on the plasma membrane of other mammalian cells, and spectrin has been identified in neurons and on membranes of intracellular organelles. A pressure field on the bilayer is generated by the conformational fluctuations of the cytoskeletal filaments to which they are anchored at their ends via anchor complexes [4] . We calculate the pressure field using a linear, flexible polymer with bulk radius of gyration Rg that is attached to a hard wall at ρ1 = (x1 , y1 ) and ρ2 = (x2 , y2 ). The pressure diverges close to the anchor points and decreases FIG. 1: Entropic pressure that a flexible, cytoskeletal protein that is anchored to a lipid bilayer with its ends exerts on the cell membrane. exponentially for large distances from the anchors, |ρ | |ρi | (i = 1, 2), see Fig. 1. The calculation of the pressure is based on the diffusion equation to calculate the polymer conformations. This implies an infinite contour length of the polymer, which is certainly a bad approximation if the distance d between the anchor points is comparable with the contour length. This situation is addressed in Ref. [5], where a Gaussian polymer model is combined with the condition of a finite contour length. Force-extension relations and end-to-end distribution functions have been calculated and the analytic theory describes experimental and simulation data very well. We use a simple argument to illustrate how a finite contour length affects the fluctuation pressure: we 2 subtract from the contour length L = 6Rg, 0 / (where = L/N is the Kuhn length of the polymer, N the number of repeat units in the chain, and Rg,0 the radius of gyration of the free chain) the anchor distance d, because this length of the chain is ’needed to connect the anchor points’ and is therefore not available for conformation fluctuations. To calculate the pressure field of the stretched chain, we simply replace Rg in the expression for the pressure by the new, effective Rg , which we obtain using 2 the effective Kuhn length, = (L − d)/N : Rg = 2 N/6 = ( /6)(L − d)2 /L. The pressure along the connection line of the anchor points is large either for very small anchor distances because of the high pressure at the anchor points or for very large an- 16 FIG. 2: Pressure field for an unstretched RBC cytoskeleton. FIG. 3: Anisotropic diffusion for a cytoskeleton that is stretched by a factor 1.5 and accordingly compressed in the perpendicular direction (for two different orientations of the hexagonal network). chor distances that are comparable with the countour length when the polymer conformation is stretched and therefore everywhere close to the lipid bilayer. Fig. 2 shows a superposition of the single-filament pressure fields for an idealized arrangement of spectrin bonds on a hexagonal lattice, as in the RBC cytoskeleton. We use a random walk and Metropolis Monte Carlo to simulate the diffusion process in the lipid bilayer. The step length is a = 1 nm, which is the typical size of the lipid molecules. The influence of the cytoskeleton is taken into account by the potential landscape that is determined for the pressure field of the cytoskeleton multiplied with the effective interaction volume for the protein under consideration. The interaction volume is determined by the size of the cytosolic part of the protein that sterically interacts with the cytoskeleton. It can be obtained either from electron microscopy data for the spectrin and the diffusing protein or phenomenologically as a fit parameter using measurements of the effective diffusion coefficient. Once the effective interaction volume is determined, we can predict several aspects of the protein diffusion [3]. For short times, the protein shows normal, fast diffusion within a single corral that is formed by the cytoskeletal bonds. Also for long times normal diffusion is observed, but with a smaller, effective diffusion constant. Long-time diffusion is hindered because the protein needs to hop across the potential barriers. The crossover between both regimes can also be observed in single-particle tracking experiments. In healthy red blood cells, the cytoskeletal filaments are usually attached at their ends but also — with a different protein complex — in their middle. In disease, the middle anchor complex can be missing. We predict that the diffusion coefficient in the disease case is by a factor 25 larger than for healthy RBCs. Furthermore, we can predict the influence of compression or stretching of the cytoskeleton on protein diffusion. For isotropic stretching, we find a decrease of the effective diffusion constant because of the increased height of the cytoskeletal barriers. For the normal RBC, the simulation results are well de- scribed by a rescaled analytic expression: the height of the potential barrier is given by the value in the middle of the bond where the cytoskeletal pressure is lowest. For anisotropic stretching, which constantly occurs in vivo when the RBC is deformed in blood flow, we predict increased diffusion along the direction of the stretch and decreased diffusion perpendicular to the stretching. In Fig. 3, we plot the relative diffusion for an anisotropically stretched network parallel and perpendicular to the direction of the stretch for different values of the effective interaction volume. For the effective interaction volume v = 6000 nm3 that applies for the band-3 protein in RBCs, we find that the diffusion is almost completely asymmetric if the RBC cytoskeleton is stretched by a factor 1.5 and accordingly compressed in the perpendicular direction (to preserve the total area, which is fixed by the lipid bilayer). Our results quantify the interaction between the cytosolic part of a transmembrane protein and a cytoskeleton that consists of a two-dimensional network of flexible polymers, as for example found in RBCs. We predict the changes in the diffusion due to lack of anchor proteins, such as a missing middle anchor complex in forms of hereditary spherocytosis. We also predict changes of the diffusion caused by a stretched cytoskeleton. [1] M. J. Saxton, Curr. Top. Membr. 48, 229 (1999). [2] M. Tomishige, Y. Sako, and A. Kusumi, J. Cell Biol. 142, 989 (1998). [3] T. Auth and N. S. Gov, Biophys. J. 96, 818 (2009). [4] T. Auth, S. A. Safran, and N. S. Gov, New J. Phys. 9, 430 (2007). [5] R. G. Winkler and P. Reinecker, Macromolecules 25, 6891 (1992). 17 Attractive colloidal rods in shear flow M. Ripoll, R. G. Winkler, G. Gompper IFF-2: Theoretical Soft-Matter and Biophysics Suspensions of rod-like colloids show in equilibrium an isotropic-nematic coexistence region, which depends on the strength of an attractive interaction between the rods. By means of hydrodynamic simulations, we study the behavior of this system in shear flow for various interaction strengths. The shear flow induces alignment in the initially isotropic phase which generates additional free volume around each rod and causes the densification of the isotropic phase at the expense of an erosion of the initially nematic phase. Furthermore, the nematic phase exhibits a collective rotational motion. The density difference between these two regions at different shear rates, allows us to determine the binodal line of the phase diagram. The results are in good agreement with experimental observations. Phase transitions occurring in soft matter systems are significantly affected by flow. Both the nature and location of the phase transition lines are changed due to the applied flow. The challenge is to find the parameters that determine the non-equilibrium steady states under flow conditions. Colloidal-rod suspensions constitute a particularly interesting system to study the effect of flow on their phase behavior, since rod orientation is strongly coupled to the shear field. Rods in the isotropic (I) phase align with the flow and become paranematic (P). This suggests that the transition to the nematic (N) phase, where rods have orientational order, is facilitated by shear. On the other hand, rods in the nematic phase undergo a collective tumbling motion in the presence of shear flow. The question that then arises is how these two effects will affect I-N detailed understanding of the flow behavior of a model system of attractive colloidal rods is useful for industrial applications where shear alignment of elongated objects, such as carbon nanotubes, wormlike micelles, and polymers, play a role. The non-equilibrium phase diagrams of attractive colloidal rods in shear flow have been investigated by mesoscale hydrodynamic simulations and compared to small-angle light-scattering (SALS) and rheology experiments [1, 2]. Earlier rheology experiments [3] have studied the non-equilibrium binodal of fd-virus dispersions under shear flow conditions for a single, FIG. 1: Snapshots of the simulation box with = 3.5, (a) at equilibrium, and (b) in a tumbling event at γ ˙ = 0.003. Colors in (a) and (b) are coding the rod orientation: horizontal is red, vertical is green and perpendicular to plane of view is blue. Red arrows in (b) denote flow direction. (c) Time evolution of the normalized density φ/φ0 , and (d) of the orientational order parameter Sx , along the gradient direction. fixed strength of attraction. This study showed that the P-N transition changes on applying flow. The simulations allow for a microscopic understanding of the behavior of coexisting phases and their interface under shear, including the possible role of collective tumbling motion of rods. The attractive rod-rod interactions are systematically varied, which affects the phase behavior, interfacial properties of coexisting phases as well as the tumbling behavior. Molecular dynamic simulations of rod-like colloids of aspect ratio 20 with attractive interactions (LennardJones potential with a minimum of , in units of the thermal energy kB T ), are combined with a mesoscopic description of the solvent known as multiparticle-collision dynamics (MPC). This hybrid approach has been shown to account for long-range 18 Dhont. Although this time is not uniquely defined. the nematic phase has a higher concentration than the isotropic phase. We believe that this is due to the higher packing fraction of rods in the nematic phase. The periodic motion of the nematic director during tumbling and kayaking is characterized by a time τ between subsequent flow-aligned states. As can be seen from Fig. [4] R. G. Rev. This agrees with the experimentally determined tumbling-to-aligning phase separation line that appears at high concentrations and high shear rates and ends at the maximum of the binodal. [1] M. and S. The factor γ ˙ max can be identified with the maximum of the binodal. The interface between the coexisting tumbling and flow aligned states is therefore highly dynamic. P. γ ˙ max and the difference in packing fractions between the isotropic and nematic phase in equilibrium. as indicated in Fig. Mussawisade. 20. vz ) = (γy. Holmqvist. This behavior is characterized by both the local concentration and orientational order parameter ˆ the local ˜ Sx ( y ) ≡ 3 u ˆx u ˆx − 1 /2. This implies that the distance between rods is smaller. we observe that the tumbling time increases with increasing interaction strength. M. Phys. where u ˆx is the component of the unit vector connecting the end-points of a rod along the flow direction. P. E 69. Shear flow orients the particles in the initially isotropic phase. i. a clear trend can be observed. all data points fall onto a master curve. K. Matt. The dashed lines are the conjectured master curve. Lettinga and J. We determine the equilibrium phase diagram as a function of the strength of an attractive interaction between the colloidal particles. Winkler. We find a widening of the two-phase coexistence region. The collective rotational motion induced by shear has been observed only in the coexisting nematic phase. J. Lettinga. K. Phys. which are identical in the actual study and in the experimental one [1]. 16. vy . as observed previously well inside the nematic region. Ripoll. Gompper. This mechanism is responsible for the reduction of the density difference between the two phases. [3] M. it leads to a collective rotation motion of large groups of rods in the nematic phase. The the coexistence region widening is the same effect as observed in equilibrium. S3929 (2004). Grelet. and therefore the hydrodynamic friction for their motion parallel to each other is larger. Winkler. the concentration is expressed in terms of ϕnem . R. 168302 (2008). 1c. Fig. Rev. We find that τ decreases linearly with the inverse shear rate. K. This generates free space around each rod and facilitates the transfer of rods from the nematic into the paranematic layer. 1c. G.: Cond. and G. E. Purdy. where the director of the nematic phase is aligned parallel to the interface. Mussawisade. Matt. More importantly. ˙ 0. Dhont. Ripoll. the proportion of the nematic phase in the equilibrium state. More interestingly. [2] M. A snapshot of I-N coexistence is shown in Fig. This is seen for all nematic domains in coexistence with paranematic regions and for all strengths of attractions studied. and G. 0). hydrodynamic interactions between rods [4].FIG. 1a. 2: Non-equilibrium phase diagram obtained from simulations for various values of the strength of attraction interaction. Matt. Gompper. Phys. 051702 (2004). Fraden. [5] Z. M. G. R. with γ ˙ the applied shear rate. Winkler. K. (vx . In Fig. With this normalization. This result very nicely agrees with recent experimental results in equilibrium [5] in which attraction is induced by a variable amount of depleting polymer. Ripoll. The simulated system is prepared in equilibrium with coexisting isotropic and nematic phases. 2 for different attractions. Furthermore. R. 1b. 404208 (2008). Dogic. K. The inset in Fig. This is in contrast to the paranematic phase that remains flow-aligned without any collective motion. Adams. 2 shows that an increasing attraction between rods broadens the coexistence region and leads to an increase of γ ˙ max . Shear flow also induces a rotational motion of individual rods. J. Gompper. G.: Cond. The shear rate is scaled by γ ˙ max . as expected. Precisely the same master curve was also extracted from experimental results. and the overline indicates averaging over the vorticity and flow directions. Phys. 1d demonstrates the periodic tumbling motion of rods in the nematic phase. which is small for weak interactions strengths. S3941 (2004). 2. P. The inset presents the unscaled data. Simulations for shear rates just above γ ˙ max indicate in fact that the system evolves into an homogeneous state. Binodals are determined at times where the density of the paranematic state has reached a stationary value and are plotted in Fig. Lett. From this striking result we conclude that the effect of attractive interactions on the non-equilibrium phase diagram is reduced to two parameters.: Cond. e. We want to emphasize that no nematic tumbling states have been found in the homogeneously nematic phase obtained for rates above the maximum of the binodal. G. and becomes very pronounced for stronger interactions. Shear is then applied with the imposed flow direction parallel to the interface. 19 . J. The time dependence of the density φ and orientational order parameter Sx of rods as a function of the position y along the gradient direction is plotted in Fig. 16. Phys. 101.d. with shear rates normalized by the maximum shear versus the fraction of equilibrium nematic phase. G. J. and M. a multi-sperm system shows swarm behavior with a power-law dependence of the average cluster size on the width of the distribution of beating frequencies. In nature. The beating tails not only propel the sperm through a fluid. In particular. The snake-like motion of the tail propels the sperm through a fluid medium very efficiently. spontaneous curvature » – cs. The higher animal sperm typically have tails with a length of several tens of micrometers.tail (x. Elgeti. which beats in a roughly sinusoidal pattern and generates forces that drive fluid motion.4π < ∆ϕ < 1. fs is the beating frequency of the s-th sperm. and A is a constant related to the beating amplitude. see Fig. ). which generates a propagating. We study the hydrodynamic interaction and cooperation of sperm embedded in a twodimensional fluid. We concluded that hydrodynamic interaction and volume exclusion play important roles in the cluster formation of healthy and motile sperm. it describes the helical motion of swimming sperm in three dimensions [4]. in mammalian reproduction. 1. We describe the motion of the surrounding fluid by using a particlebased mesoscopic simulation method called multiparticle collision dynamics (MPC) [2.tail + A sin −2πfs t + qx + ϕs . viscous forces dominate over inertial forces. On this scale. Before they find the ova. G. and L0 is the tail length.tail determines the average spontaneous curvature of the tail. 3]. the hydrodynamic interaction and volume exclusion are not negligible.5π . Simulation data are shown for fixed phase difference (red.5% difference in the beat frequencies of the two sperm (solid line). The constant c0. For example. With these hydrodynamic effects. sinusoidal wave along the tail. Gompper IFF-2: Theoretical Soft-Matter and Biophysics Sperm swimming at low Reynolds number have strong hydrodynamic interactions when their concentration is high in vivo or near substrates in vitro. the swimming motion of a sperm corresponds to the regime of low Reynolds number. the local density of sperm is sometimes extremely high. sperm clusters are always seen after the system has reached a stationary state. t) = c0. This simulation method has been shown to capture the hydrodynamics and flow behavior of complex fluids over a wide range of Reynolds numbers very well [3]. The wave number q = 4π/L0 is chosen to mimic the tail shape of sea-urchin sperm. sperm have to overcome many obstacles in their way. The distance dh is also shown as a function of time t (top axis) in a simulation with a 0. which show much stronger thrust forces than a single sperm. We also found that the average cluster size has a powerlaw dependance on the width of beat frequency distribution [5].Cooperation of sperm in two dimensions: synchronization and aggregation through hydrodynamic interactions Y. The sperm model consists of particles connected by stiff springs. The interpolating (red) line is a linear fit for 0. 20 . Thus. the average number of sperm per ejaculate is tens to hundreds of millions. Here. comparable to the length of their flagellum. In experiments with rodent sperm at high densities [1]. synchronization and attraction. Two effects of hydrodynamic interaction are found. FIG. Yang. In our simulations. We analyzed the hydrodynamic interaction between two sperm and the aggregation behavior of multisperm system. At this length scale. but also create flow fields through which sperm interact with each other. ϕs is the initial phase of the s-th sperm. so that the average distance between sperm is on the scale of ten micrometers. we construct a coarse-grained sperm model in two dimensions. 1: Head-head distance dh of two cooperating sperm. E. Sperm motility is important for the reproduction of animals. The beating motion is determined by a time-dependent. A healthy mature sperm of a higher animal species usually has a flagellar tail. The sperm seem to take advantage of strong interactions with neighbor cells of the same species to win the fertilization competition. which form a circular head and a filament-like tail. motile clusters consisting of hundreds of cells have been found. Yang. This “attraction" process takes much longer time then synchronization. we perform simulations with Gaussian-distributed beating frequencies. Ihle. Malevanets and R. 3 as a function of the width δf of the frequency distribution. D. there is a balance between cluster formation and break-up when δf > 0. 39 of NIC series. At the same time. Chem. Considering that in real biological systems the beat frequency is not necessary the same for all sperm. Fig. Once a system reaches a stationary state. Sci. [1] S. The motile sperm embedded in a two-dimensional fluid aggregate through hydrodynamic interactions and volume exclusion. Then. 1 (2009). Kapral. Breed and T. Thus. With the knowledge of hydrodynamic interaction between two sperm. Kroll. Fig. [2] A. since the phase difference to other cells in the cluster increases in time due to the different beat frequencies. [5] Y. pp. Adv. 168701 (2004). the possibility to bump into another cluster decreases. and f = 1/120 is the average frequency. Münster.12 δf (upper) and < nc >= 1. PLos One 2. A possible way of break-up is by bumping head-on into another cluster. Gompper. Gompper. they synchronize their tail beats by adjusting their relative position. Aldana. Rev. E 78. The lines indicate the power-law decays −0. (∆f )2 is the mean square deviation of the frequency distribution. NIC proceedings. Gompper. Generally.9% of a Gaussian distribution of beating frequencies. −γ < nc >∼ δf (1) with γ = 0.196 < nc >= 2. < nc >. the cluster size can grow by collecting nearby free sperm or by merging with other clusters. δf = (∆f )2 1/2 / f denotes the width of the Gaussian frequency distribution. The negative exponent indicates that the cluster size diverges when δf → 0. it decreases with decreasing sperm density or increasing δf . FIG. [3] G. it does not disintegrate without a strong external force. Lett. we found two effects of the hydrodynamic interaction. Elgeti and G. When two sperm are close in space and swimming parallel. G. H.01.5π . Phys.201 −0. on the width of the frequency distribution δf . Elgeti. As the cluster size increases. edited by G. 221.20±0. Polym. Large clusters of synchronized and tightly packed sperm are formed. J. We give each sperm random initial position and orientation. The average cluster 21 . W. Moore. J. Similar power-law behaviors have been found in simulation studies of swarm behavior of self-propelled particles [6]. Birkhead. T. M. 1 shows the linear relation between phase difference and headhead distance of a sperm pair.9%. once a cluster has formed. Vol. we study multi-sperm systems. Here. Kremer. 8605 (1999). [6] C. Winkler. Data are shown for a 50-sperm system ( ) and a 25sperm system (◦). size always increases for δf = 0. Note that we employ periodic boundary conditions. 061903 (2008). D. The black frames show the simulation boxes. The cooperation of our sperm model fails when the phase difference ∆ϕ > 1. and R. Phys. motile clusters. 110. thus the rate of break-up decreases. If δf = 0. 2 shows a snapshot of a 50-sperm system with δf = 0. e170 (2007). 2: A snapshot from simulation of 50 sperm with widths δf = 0. Immler. Rev.55 δf (lower). M. Wolf. sperm cells can leave a cluster after sufficiently long time. [4] J. 53-61 (2008). Phys. Two sperm stay locked in phase and swim together until their phase difference becomes too large. For δf > 0. synchronization and attraction. This “synchronization" process is accomplished in a very short time. the synchronized sperm have a tendency to get close and form a tight pair. and M. Synchronization and attraction effects help the sperm to form large. 3: Dependence of the average stationary cluster size. This coordinating behavior decreases the total energy consumption of two sperm.In two-sperm simulations. The red ellipses indicate large sperm clusters. D. and G. Huepe and M. G. R. however. 92. The average cluster size < nc > is plotted in Fig. FIG. the cluster-size distribution function obeys a power law. Figure 3 also shows a power law decay. Schwarz P . For example. The interfacial free energy per unit area of a grain boundary depends on the angles with respect to the crystalline axes. The calculations are performed on a discrete threedimensional real-space lattice φijk .the unit cell is large. of the order of 10 nm. 5] with a single scalar order parameter φ(r) which describes the local oil-water concentration difference. FIG. gyroid. The interfacial free energy per unit area is found to be very small. which are encountered in physical systems. The geometrical properties of the grain boundaries are evaluated on the isosurface φ(r) ≡ 0 which defines the position of the surfactant monolayer. Initially. diamond and Schwarz P phases. Properties of cubic surfactant phases have been a subject of extensive theoretical and experimental interest. Examples of such phases include the gyroid G. as templates for mesoporous systems and for the crystallization of membrane proteins. The lattice constant which corresponds to the dimensions of the fundamental building block . Belushkin. 22 . 1: Configuration of the surfactant monolayer for the lamellar phase at a twist angle of 53o (left) and the gyroid phase at a twist angle of 90o (right). G. The free energy is minimized using a method based on the gradient descent algorithm [6]. whilst tilt grain boundaries in the lamellar phase have been shown to be omegashaped at large tilt angles and chevron-shaped at small tilt angles [3]. Amphiphilic molecules added to an immiscible oilwater system self-assemble into a large variety of structures. During the nucleation of a cubic phase. The calculations are based on a Ginzburg-Landau theory of ternary amphiphilic systems [4. twist grain boundaries in the lamellar phase are well described by Scherk’s minimal surfaces at large twist angles and are helicoid-shaped at small twist angles [1. Gompper IFF-2: Theoretical Soft-Matter and Biophysics Cubic surfactant phases are mesoscale liquidcrystalline structures in which the surfactant monolayer separating the oil-rich and waterrich domains often has a triply-periodic minimalsurface geometry where the mean curvature H vanishes on the whole surface. therefore the density of grain boundaries should be high in these surfactant phases. We have investigated twist grain boundaries in the lamellar. and between two grains of the same ordered phase which differ by their spatial orientation.Twist grain boundaries in cubic surfactant phases M. 2]. Schoen I − W P . between ordered and disordered phases. Phases with cubic symmetry often feature a triply-periodic minimal surface (TPMS) configuration of the surfactant monolayer. 1. F − RD. The structure of the monolayer in the grain boundaries is found to be very close to a minimal-surface geometry. half of each calculation box is filled with a phase rotated by an angle −α/2 and the other half with a phase rotated by an angle +α/2. Neovius C (P ) and others. diamond D. many classes of defects arise. In amphiphilic systems many kinds of interfaces occur: between two ordered phases. with applications in biological systems. Configurations of the surfactant monolayer in the full simulation boxes of the lamellar phase at a twist angle of 53o and the gyroid phase at a twist angle of 90o are shown on Fig. The grain boundaries are located in the middle and at the top/bottom of each box. We report here on the investigation of twist grain boundaries in cubic surfactant phases. It is determined using the Ginzburg-Landau theory and a complementary geometrical approach based on the Canham-Helfrich curvature Hamiltonian. Experiments on block copolymer systems have revealed the structure of many interfaces. . 6502 (2000) [4] G. In the lamellar. Schick.8o . The excess free energy of the grain boundaries exhibits a non-monotonous dependence on the twist angle α. Gaussian-curvature distributions (Fig. Cohen et al. J. Lett. 2: Twist grain boundary geometries for the lamellar (top). The bulk-phase regions of the Schwarz P phase are greatly affected by the presence of grain boundaries. gyroid (middle) and diamond (bottom) phases for twist angles of 53o (left) and 90o (right). Therefore. 134712 (2009) FIG. Macromolecules 26. Gompper. gyroid and diamond phases the thickness of the grain-boundary regions is about one unit cell of the bulk-phase regions. gyroid G. Geometrical approaches based on the Canham-Helfrich curvature energy Hamiltonian yield similar results [6]. [1] S. 1116 (1990) [5] U. therefore the configurations of the surfactant monolayer are good approximations of minimal surfaces [6]. The black vertically-shaded and red slant-shaded histograms correspond to bulk and grain-boundary regions. S.The locations of the grain boundaries are determined quantitatively by relating the inital and final simulation boxes [6]. 3) show that [6] • the Gaussian curvature distributions in the bulkphase regions are consistent with the exact results obtained from the Weierstrass representation • in the Lα . Macromolecules 39. Rev. and the negative values for the P phase show it to be unstable with respect to the nucleation of grain boundaries at the investigated point in the phase diagram. Phys. thus grain boundaries preserve the typical length scales of the bulk phases. Jinnai et al. Squared-mean and Gaussian curvature distributions are calculated for each phase and twist angle. diamond D and Schwarz P surfactant phases as extracted from Ginzburg-Landau theory.. The free energy of the grain boundaries is very small. The difference in the grain-boundary free energy and the free energy of bulk-phase regions of equal volume is of the order of 1% at the maxima of Fig. 130. M. Gido et al. 4. respectively. The surfactant monolayers in grain-boundary regions of the lamellar (top). Phys. the density of grain boundaries should be high in these surfactant phases. 65. The blue solid line corresponds to the exact result obtained from the Weierstrass representation. Chem. FIG. 4: Grain-boundary excess free energy per unit area as a function of the twist angle α for the lamellar. Belushkin. 3: Gaussian-curvature distribution for the G phase at a twist angle α = 27. E 59. 23 .. gyroid (middle) and diamond (bottom) phases are shown on Fig. 5528 (1999) [6] M. G. 2 for twist angles of 53o (left) and 90o (right). Macromolecules 33. 5815 (2006) [3] Y. G and D phases the geometry of the grain boundaries is significantly different from the geometry of the bulk phases Excess free energy per unit area of the grain boundaries determined from Ginzburg-Landau theory as a function of the twist angle α is shown on Fig. Phys. Gompper. Gompper. G. Squared-mean-curvature distributions show that the mean curvature of the surfactant monolayer essentially vanishes both in the bulk-phase and grainboundary regions. 4506 (1993) [2] H. Schwarz. Rev. FIG. 4. the faster flow in the center of the capillary and the slower flow near the walls in- duces this shape change.6 µm and 2 µm with constant area and volume. optical microscopy of micro vessels [1] have shown that individual RBCs can adopt a parachute shape. The RBC deformability is reduced in blood related diseases. At thermal equilibrium. At very high flow velocities and very low HT . the discocyte shape of a RBC can be predicted theoretically by minimizing the membrane’s bending and stretching energy subject to a fixed surface area and volume. As the RBC became more rigid. The RBC membrane is treated by a discretized mechanical model of a two-dimensional elastic membrane parametrized by κ and µ.Clustering and alignment of red blood cells in microcapillaries J. We consider a number nves of vesicles in capillary tube segments of length Lz and radius Rcap with periodic boundary conditions in the flow direction. This transition was accompanied by a sudden drop in the pressure needed to drive the flow at a given velocity. The full length of the cylindrical simulation tube is Lz . aggregation. We study the collective flow behavior of many RBCs. that is. We have already studied the shape changes of a single. where the capillary diameter is comparable to the diameter of the RBCs.7 and nves = 6. The bilayer’s resistance to a bend is controlled by a curvature energy with a bending rigidity. a faster the blood flow was needed to achieve this shape transition. nonequilibrium conditions. the deformability of a RBC is important for the regulation of oxygen delivery. However. a dynamically triangulated surface model [3]. Here L∗ ves = Lz /nves Rcap 24 . HT ). isolated RBC in a blood flow at high dilution [2] using a mesoscopic simulation technique which combines two methods. G.3 or HT = 0. At high HT . and the flow velocity of the RBC suspension. H. The critical flow velocity associated with the shape transition from a discocyte at low velocities to a parachute at higher ∗ ∼ 5. The spectrin network enables an RBC to remain intact while changing its shape in blood flow through narrow capillaries with diameters of 0. Therefore. 1: Sequential simulation snapshots of six RBCs in a dilute blood suspension (L∗ ves = 3.28/L∗ ves ) at v0 = 7. McWhirter. The main focus of the present work is to study the collective flow behavior of many interacting RBCs at very low and high HT [4. In the absence of flow.2 µm to 10 µm. At high dilution in fast blood flows through narrow capillaries. Two essential control parameters in our study are the volume fraction of RBCs (the tube hematocrit. which acts as a cytoskeleton and is responsible for the shear elasticity of the membrane. κ. particle-based dynamics called Multi-particle Collision Dynamics (MPC). The solvent hydrodynamics is described by an explicit. and alignment of red blood cells (RBCs) in cylindrical capillary flow is investigated by mesoscopic hydrodynamic simulations. under flowing. µ. the parachute-shaped RBCs can be found in clusters that are stabilized by the hydrodynamic solvent flows. L. Vves . the RBCs can exhibit one disordered phase and two distinct ordered phases depending on HT and the flow velocity. Noguchi. and the spectrin’s resistance to a shear strain is characterized by a shear modulus. coupled to hydrodynamic flows are important contributors to the RBC morphology. Our studies showed that the shape change of an RBC from a discocyte to a parachute occurs at a critical flow velocity which depends on the material parameters κ and µ [2].084 = 2 ∗ nves Vves /πLz Rcap = 0. FIG. Gompper IFF-2: Theoretical Soft-Matter and Biophysics The shapes. such as diabetes mellitus and sickle cell anemia. Thermal fluctuations. included in the simulation method. with these diseases the resistance to flow is relatively high and the heart must work harder to produce the higher pumping pressures needed to ensure normal blood flow. The RBC membrane consists of a lipid bilayer supported by an attached spectrin network. Z . Top panel shows a 6 RBC cluster while velocities is vc the bottom two show the break-up of this cluster at a later time. the shape adopted by the RBCs is determined by the competition between these mechanical properties and the external hydrodynamic flow forces arising from the blood plasma (the solvent) suspending the RBCs. human RBCs have a biconcave-disc (discocyte) shape whose maximum diameter and thickness are 7. 5]. spherical white blood cells. ∗ and v0 ≡ v0 τ /Rcap . interdigitated parallel rows. [5] P. The hematocrit varies between HT = 0. Gompper. At very low HT . 49 (1996).875 and v0 = 2. as shown in Fig. Second. 106. 102. In summary. ∗ and (c) Zigzag-slipper phase for L∗ ves = 0. the aligned-parachute (P) and zig-zag slipper (S) phases (Fig. Natl. 3: (a) Phase behavior as a function of average vesicle ∗ distance L∗ ves and mean flow velocity v0 . At high HT . 6039 (2009). (b) Pressure drop ∆Pdrp per vesicle for the aligned-parachute phase (simulations with nves = 1) and the zigzag-slipper phase (simulations with nves = 6) at the same volume fraction (L∗ ves = 0. McWhirter. Microcirculation 3. or resistance to flow. the RBCs appear as discocytes with random orientations and no significant long-range spatial cor∗ relations. N. Symbols represent the disordereddiscocyte ( ).28/L∗ ves . There are two reasons for the cluster formation. Noguchi and G.45. Noguchi. Suzuki. However.H. C. since HT = 0. leading to cluster break-up events as shown in Fig. the collective behavior of several RBCs determines their flowinduced morphology and resistance to flow. the S phase ∗ produces a larger pressure drop ∆Pdrp . Acad.75.85 and low ves ∗ v0 . Proc. ∗ (b) Aligned-parachute phase for L∗ = 0 . and N. these fluctuations have the effect of moving an RBC off the capillary axis to regions of slower flow. [2] H. 14159 (2005). Soutani. this single RBC approaches the cluster. the S phase occurred unexpectedly at smaller L∗ ves . Future studies will examine the dependence of flow properties on channel geometry. [4] J. [3] H. 799 (1980). one is disordered. Blood Cells 6.85 is destabives lized by the thermal fluctuations that are incorporated into the mesoscopic simulation method. and the introduction of more rigid. Phys. Noguchi and G. Sci. three distinct RBC ‘phases’ exist (Fig. respectively. therefore. Natl. we have shown that at very low RBC volume fractions the RBC deformability implies a flowinduced cluster formation at high blood flow velocities. Proc. 875 and v ves 0 = 10. Sci. 3(b).37). K. while the remaining two are ordered. As v0 approaches vc .22 and HT = 0. or their incorporation is not as simple as in MPC. and G. M. E 72. and zigzag-slipper (•) ∗ phases. fluctuations in the RBC shape increase. forming a larger cluster. Gompper. for nves = 6. thermal fluctuations are not incorporated. The P phase was expected at higher v0 based on the simulations examining the shape transition to a parachute of a single. Gaehtgens.FIG. increas∗ ∗ ing its lifetime. 011901 (2005). polydispersity of the RBC suspension. given these earlier simulations. where the characteristic shape 3 relaxation time is τ = η0 Rcap /κ. FIG. H. the effective hydrodynamic flow mediated attractions between the RBCs in a cluster stabilize the cluster. aligned-parachute ( ). the disordered-discocyte (D) phase. 3). [1] Y. A gravitation force mg is used to generate flow along the Z axis where 2 v0 = mns gRcap /8η0 and η0 is the viscosity of the suspending solvent. at high volume fractions. associated with the discocyte to parachute transition. Tateishi. 1. isolated RBC under flow [2].75 and v0 = 10. Acad. slipper-shaped RBCs form two regular. Albrecht. 2: Simulation snapshots of nves = 6 RBCs: (a) ∗ Disordered-discocyte phase for L∗ ves = 0. Du ¨hrssen. vc . In other theoretical approaches. In addition. bending closer to the capillary axis or center where the flow is fastest. corresponding to HT = 0.L. In the D phase at L∗ 0. than the periodic P phase under equivalent conditions. Rev.5. Maeda. parachute shaped RBCs form sta∗ ble clusters at flow velocities v0 much higher than ∗ the critical flow velocity. a single RBC separated from a neighbouring cluster of RBCs is more deformed by the flow than its neighbours. 2). Gompper. Curiously. 25 . Here. An alignedparachute conformation at L∗ 0. First. 26 . In this position it operates several neutron scattering instruments at the research reactor FRM II in Munich. Dieter Richter The Institute for Neutron Scattering is concerned with neutron research placing major emphasis on soft condensed matter. i. The institute has modern chemical laboratories for the synthesis. impedance spectroscopy. A major part of the Soft Matter studies is done on polymers. characterisation.g. aggregates). 27 .g. These polymers often have a complex architecture (copolymers. calorimetry. Apart from their structure.) to tailor them for industrial applications. x-ray scattering.e. materials that react strongly to weak forces. light scattering. and modification of Soft Matter. Another field of interest are complex liquids such as microemulsions or colloid systems. In order to complement neutron scattering experiments several ancillary techniques are used in the institute: rheology.and timescales. The Institute for Neutron Scattering is partner in the Jülich Centre for Neutron Science JCNS. at the Institut Laue-Langevin in Grenoble. Another focus of research is the development of neutron instrumentation for research reactors and future spallation sources worldwide. Neutron scattering is a valuable tool for these systems because it reveals structure and dynamics of Soft Matter on the relevant length. These instruments are available to guest researchers on request. USA. proteins) are studied concerning their structure and dynamics. and computer simulation. star-polymers etc. Biological materials (e.IFF-5: Neutron Scattering Director: Prof. we are interested in the dynamics of polymers in melts and solutions (e. gels. rubbery networks. and at the Spallation Neutron Source in Oak Ridge. poly(1. In agreement with this result the molecular weight distribution is elevated and the measured molecular weight is about 25% smaller than the calculated molecular weight. The SEC trace of sample PBO-1 is given in Figure 2. 1: Structures of different alkylene oxide monomers and polymers.2octylene oxide) (POO) and even higher PAO were obtained by polymerizing the corresponding monomers with zinc organic catalysts [4-6]. however. This measure allowed eliminating by-products almost completely in the synthesis of poly(1. the polyalkylene oxide family offers interesting possibilities with respect to their rheological properties due to the systematic variability of the polymer chain diameters. and poly(1. As a result high and low molecular weight by-products are formed [2. Beside the main peak the signal contains a low molecular weight tailing and a high molecular weight shoulder at higher and lower elution volumes.2-hexylene oxide). The reason for the side reactions is the relatively high acidity of the methyl protons of PO leading to different types of termination and chain transfer reactions. For polymers synthesized above room temperature significant amounts of byproducts were found. defined by the amounts of monomer and initiator introduced into the reactor. poly(1. This leads to large amounts of byproducts. 3]. The most widely employed technique for the polymerization of alkylene oxides is anionic ring-opening polymerization using sodium or potassium alcoholates as initiators. Poly(1. yields polymers with broad MWD and does not allow producing block copolymers.2-butylene oxide). In a first series of experiments butene oxide was polymerized at different temperatures and using different solvents and initiators. In the older literature.2octylene oxide). In this experiment potassium tert.2-butylene oxide) (PBO). monomer O H2C CHR polymer ( CH2 CH O ) R=H R=CH3 R=CH2-CH3 R=(CH2)3-CH3 R=(CH2)5-CH3 ethylene oxide propylene oxide 1-butylene oxide 1-hexylene oxide 1-octylene oxide R polyethylene oxide (PEO) polypropylene oxide (PPO) polybutylene oxide (PBO) polyhexylene oxide (PHO) polyoctylene oxide (POO) FIG. poly(1. the amphiphilicity of the resulting block copolymers can be varied systematically. Because of the dipole moment along the chain PAO represents an alternative to the widely used polyisoprene for dielectric measurements. In this work the anionic polymerization of hydrophobic alkylene oxides was investigated at different temperatures. The new synthetic method is generally of interest due to the rheological and dielectric properties of the polymers and the possibility to tune amphiphilicity of the amphiphilic block copolymers. Figure 1 illustrates the chemical structures of some polyalkylene oxides and the corresponding monomers. Allgaier IFF-5: Neutron Scattering The anionic ring-opening polymerization of alkylene oxides like propylene or butylene oxide is accompanied by strong side reactions. The lowest members of the polyalkylene oxide (PAO) family. In addition. For the polymerization of propylene oxide (PO) strong side reactions are present. In contrast to most other block copolymers the hydrophobic moieties in the PAO block copolymers are soluble in a large variety of oils. was 15. are known for a long time. obtained from the amounts of initiator and polymerized 28 . by combining PEO with the other PAO. solvents and initiating systems [1]. This technique allows to polymerize ethylene oxide (EO) basically free of side reactions. With the help of crown ethers the polymerization temperature could be reduced to -23°C. Furthermore the method was employed to synthesize amphiphilic block copolymers of these polyalkylene oxides with polyethylene oxide as the hydrophilic moiety.Synthesis of Polyalkylene Oxide Homo. The target molecular weight.and Block Copolymers J.-butanolate (KOt-Bu) was used as initiator at 80oC and toluene was chosen as an inert solvent.000. In contrast to the limited availability of the higher members. there are reports dealing with the synthesis of the higher and more hydrophobic PAO than PPO. polyethylene oxide (PEO) and polypropylene oxide (PPO). making them ideal candidates as additives in microemulsion systems. This technique.2-hexylene oxide) (PHO). 000 could be obtained without significant by-product content and low molecular weight distributions. In further experiments other alkali metal initiators and more polar aprotic solvents were tested in order to reduce the polymerization temperature without reducing reactivity and extending the polymerization time too much. Schick . 60. B 1967. M.000 were basically free of homopolymer. Marcel Dekker: New York. V. the target molecular weight was increased from 15. J. Using this system the polymerization temperature could be reduced o below -20 C and within experimental error no byproduct could be detected (see Figure 2. J. However. Polymer 1970.monomer. 2: SEC refractive index signals of PBO samples. Again different alkali metal initiators in combination with different crown ethers were tested. Koleske. Vol.. [4] Lal. Especially in less polar solvents they increase the degree of ion-pair separation. Even block copolymers having block molecular weights of 50. Nace. Vol. 3: SEC refractive index signals of a PBO-PEO block copolymer and the corresponding PBO homopolymer with block molecular weights of approximately 10. [3] Whitmarsh R. Frederick. S. In Alkylene oxides and their polymers. 35. These additives are strong complexing agents for alkali metal ions. Chapter 4. Sci. Because of the reduced level of byproducts already achieved without crown ethers. T. and POO as hydrophobic blocks and PEO as hydrophilic block were produced using the polymerization techniques described before. C. PHO. Sci. 40.. Therefore a different strategy was chosen based on crown ethers. 11. Wilbold. 5. Chang. This again underlines that the new low-temperature method using crown ethers yields model polymers of high structural quality. Chapter 1.. A-1 1966.000 to 50. 793. However it was necessary to purify those monomers by careful fractional distillation due to the lower purity of the commercial raw material compared to butene oxide. A-B block copolymers containing PBO. R.000. 518. F. J. PBO-PEO PBO P BO-1 P BO-15 34 elution volume 40 4 0 FIG. [2] Bailey. 626.. This technique allows to synthesize the block copolymers by successively polymerizing the hydrophobic alkylene oxide and EO in a one-potreaction. [1] Allgaier. It shows a symmetrical signal. As an example the SEC trace of a PBO-PEO block copolymer having block molecular weights of 10.. at 40oC still significant amounts of by-product were present and at lower temperatures polymerization was extremely slow. Ed. J.. M. This measure generally increases the relative fraction of by-products and helps to get a clearer picture about improvements of the product quality. J. E. The more hydrophobic monomers hexene oxide and octene oxide could be polymerized under similar conditions as butene oxide. Surfactant Science Series. V. Polym. 2007. Within experimental error the calculated compositions and the measured ones were similar. In our case it was possible to reduce the polymerization temperature considerably below room temperature. J. In Nonionic Surfactants – Polyoxyalkylene Block Copolymers.000 is given in Figure 3. Polym. M. Orme. Macromolecules. Surfactant Science Series. Eds. 1996. With this additional measure molecular weight of 50. F. [5] Lal.. The small values of Mw/Mn indicated narrowly distributed polymers. This measure was successful insofar the by-product content could be reduced. It turned out that KOt-Bu together with the crown ether 18C6 was the best combination. Marcel Dekker: New York. [6] Booth.000. H. 1991.. 29 . 35 40 elution volume 35 40 elution volume FIG. 4 .. At higher elution times there is no hint for homopolymer.000 to 100. In case of alkylene oxide polymerization this leads to increasing reactivities and polymerization rates. sample PBO-15). J. polymerized without crown ether at 80°C in toluene (PBO-1) and with crown ether at -20°C in toluene (PBO15). FIG. The arrow indicates schematically the movement of the active domain here without a bound cofactor. Neutron scattering provides the possibility to investigate the timescale of these large scale movements in biomolecules. Sometimes the binding of the cofactor or substrate is responsible for the formation of the right shape of the pocket. The measurements were done in dilute D2O buffer solutions. Dieter Richter1 1 2 IFF-5: Neutron Scattering IBN-4: Biomechanics 3 Institut Laue Langevin. the cofactor and the product of the reaction will be released. There must be movements of atoms. Scattering at the atomic nuclei of the protein will produce a characteristic interference pattern. opens and closes the cleft by thermal movements. The blue monomer is in the closed conformation with a NAD cofactor (orange) and an ethanol (red) in the cleft between binding domain and the active domain. It produces ethanol in yeast or converts it back to acetaldehyde in the human liver. Michael Monkenbusch1. Many transformation processes are underway in characteristic shaped bags. Peter Falus3. This effect is so far mainly known from structural studies of crystallized proteins. This “induced fit” includes small changes of single bonds but also large domain motions to prepare the active atomic configuration. 1: Tetrameric Alcohol Dehydrogenase of yeast is build up from two dimers (green-blue and gray). Bernd Hoffmann2. Sylvain Prévost4. A functional important molecule (cofactor) Nicotinamide Adenine Dinucleotide (NAD) and a substrate molecule (ethanol or acetaldehyde) bind to the binding domain in a cleft between active and binding domain. in which only certain substances fit. The second dimer (gray) is bound at the backside of the first dimer. It provides information on the location and movement of atoms. The collective motion of domains as revealed by their coherent formfactor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the cofactor to the active center. with a catalytic Zinc atom in the active centre. close to the natural environment of the protein. transport. Neutron spin echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase [1]. Grenoble.Protein Domain Motions observed in Space and Time by NSE Ralf Biehl1. 30 . Germany To bind a cofactor to the active center of a protein often a structural change is mandatory. Internal movements could also be observed by fluorescent labelling of two points on a protein with molecular biological methods to track distance changes [2]. Further information is attainable only by computer simulations. Figure 1). Again opened. they are in every cell of our body tirelessly active. which represent snapshots of different configurations. without destroying the sample. as a key fits to the lock. D2O offers the possibility to focus on the protein scattering. synthesize. As nanomachines of metabolism. France 4 Hahn Meitner Institut. and another hydrogen atom is released. Through this "induced fit" the lock for a certain key is arranged to fit. The active domain. One of the most studied proteins and one of the key enzymes is Alcohol Dehydrogenase (ADH. The closing places the active centre in the right position for the transfer of a hydrogen atom between the substrate and cofactor molecule. The shape of these pockets is determined by the sequence and three-dimensional arrangement of amino acids. Berlin. Proteins are the molecular machinery of life. divide and transform substances. because the D2O is only a weak scatterer for neutrons compared to H2O or the protons in the protein. Rudolf Merkel2. In the cleft between both domains of the open configuration (green monomer) the catalytic Zinc atom at the catalytic centre is visible. amino acids or entire domains to bring the atoms in the right configuration to get active. Movements of the atoms during the scattering process change the speed of the neutrons in the scattered beam. The change in speed for the scattered neutrons is very small. In order to detect it, NSE spectroscopy uses the Larmor precession of the neutron spins in a magnetic field as a stopwatch. The result is a length scale dependent flexibility, which differs from the expected motion of a rigid protein, only showing translation and rotation movements of diffusion. These length scale variations reflect the pattern of the movements due to the underlying dynamics. these motions as small variation of the rigid protein structure enables the calculation of the change in the effective diffusion due to overdamped normal mode motions. The change in the effective diffusion of the most prominent normal modes 7 and 11 is shown in Figure 3. Other modes show a smaller contribution. Applying the method to the protein with and without cofactor shows the influence of the cofactor onto these characteristic motional patterns. The peak structure is comparable to the measured peak in figure 2 and shows also the effect of a shift due to the bound cofactor. FIG. 2: a, Single tetramer diffusion coefficient D0eff after corrections with and without bound cofactor. The black solid line represents the calculated effective diffusion coefficients for the ADH crystal structure, including translational and rotational diffusion of the stiff protein. At low q we observe only translational diffusion. The increase with the peak structure is due to rotational diffusion, which is only visible at larger q. b, Difference of the corrected diffusion coefficients and the calculated translational/rotational diffusion coefficient, reflecting the internal dynamics. FIG. 3: Change of the effective diffusion due to movements along normal mode 7 and 11. Modes below 7 are trivial modes from translation and rotation. Without cofactor mode 7 is a mode which opens the cleft between catalytic domain and binding domain as indicated in figure 1. Mode 11 is a large amplitude mode described as torsion inside of the dimer along their main axis. The normal modes are numbered in sequence starting with the slowest timescale. The NSE spectroscopy always measures the collective movement of all protein atoms, so in the evaluation the contribution of translational and rotational diffusion to the observed scattering has to be separated from the internal dynamics (Figure 2). After this we find a remarkable peak in the difference to the rigid body diffusion shown in Figure 2b. The internal motions of a protein are quite complex. One way to explore possible large scale motions in a protein is the analysis of elastic normal modes. This method uses a simplification, it assumes only elastic forces between a reduced number of atoms representing the main structure of the protein. The method is like building a simplified model of the protein with the same shape made from elastic rubber. By shaking the rubber model it is possible to observe large scale motions with weak parts as flexible hinges between more compact stiffer domains. Analysing We found in our studies that the patterns of the movements observed are mainly due to the movement of the active domain relative to the binding domain at a timescale of 30 ns. Figure 1 illustrates schematically the movement of the active domain, leading to the opening of the gap according to normal mode 7. If the cofactor binds, it stiffens the protein, and the domain movement is significantly reduced. In ADH, we find an average amplitude of about 0.8 nm for the extent of the spatial movements between the two domains. Also we determined a spring constant of about 5 pN/nm for the stiffness of the cleft opening. [1] Ralf Biehl, Bernd Hoffmann, Michael Monkenbusch, Peter Falus, Sylvain Prévost, Rudolf Merkel and Dieter Richter Phys. Rev. Lett. 101 (2008) 138102 [2] D. W. Pistona, G.-J. Kremers Trends in Biochemical Sciences, 32 (2007) 407 31 Unexpected power-law stress relaxation of entangled ring polymers W. Pyckhout-Hintzen1 , D. Richter1 , D. Vlassopoulos2 , M. Rubinstein3 1 2 3 IFF-5: Neutron Scattering FORTH, Institute of Electronic Structure and Laser, Heraklion, Crete,Greece University of North Carolina, Department of Chemistry, Chapel Hill,USA Linear and long-chain branched polymers relax by reptation processes out of the confining tube or via arm retraction respectively. Both mechanisms are intimately related to the presence of chain end material. It is of major interest how entangled ring polymers which lack this basic ingredient of chain ends proceed to relax stress. Self-similar dynamics yielding a power-law stress relaxation is reported on model ring polymers. Here, the importance of some linear impurities becomes evident. The combination of neutron scattering and rheology for rings is a promising tool therefore to unravel details of their dynamics and their difference with linear chains. The investigation of polymer dynamics is always closely related to the particular structure or even the architecture that the polymers adopt. Fig 1a represent a reptational-like motion through an obstacle field of chains, which is clearly different from star fluctuations in Fig 1b where only arm retraction modes can relax the stress. Rings in Fig 1c and 1d may occur in double-fold shape or even behave like latticeanimals respectively. Latter has a very strong similarity with a random cayley tree (Fig 1e) for which the relaxation time spectrum corresponds to long logarithmically-spaced time scales for each of the different chain sections. These follow a hierarchical scheme and relax as is well known from former works from the outside-inwards in a sequential way, leading to typical patterns in the complex relaxation modulus. For model-branched architectures, the number of generations is coupled to the number of loss peaks. It is, however, not obvious how a ring-like polymer will perform. Fig f depicts e.g. self-interpenetration and blockage of the rings. In the former considerations the influence of linear contaminants which are the product of non-perfect linking chemistry or degradation of closed cycles to open linear chains, was not yet included. To verify theories which focus on the special structuredynamics associated with the odd architecture, it is a prerequisite to purify the rings. Recently, developments of liquid chromatography at the critical condition have shown to efficiently separate rings and linear chains via the compensation of entropic size exclusion and enthalpic interactions with the pores. The polymers so obtained were analyzed by rheology and SANS in order to assure their unchanged, original closed structure. The linear rheology for the purified rings is very compatible to the model of self-similar lattice animals of Fig 1(d,e). The associated exponentially decreasing relaxation modulus G(t) is then given as „ G( t ) = GN 0 t τe «− 2 5 „ exp − t τring « FIG. 1: Different correlations of structure with assumed dynamics are summarized and explained in the text. The comparison of ring with branched polymers is made clear. This is distinctly different in linear or branched polymers which show an extended entanglement plateau. This expression for the relaxation modulus already includes constraint-release effects for loop rearrangement as well as dynamic dilution and contains no further adjustable parameters. SANS measurements are shown in Fig 2. From dilute solution and in a melt of linear chains (not shown), unperturbed chain dimensions as well as the swelling of the ring by excluded-volume statistics could be 32 confirmed. The experimental slopes in Fig 2 are typical. Very good agreement in both size as well as the scattering amplitudes was obtained. The ring in good solvent is swollen by a factor of 1.5 which is in accordance with estimates of about 1.4 in literature. No traces of impurities of linear chain could be spotted from SANS. We have forwarded in Fig 3 the molecular picture that linear chains must be bridged at this extremely low concentration by the rings to be so effective and therefore provide considerable increase in the melt viscosity. The entropically driven penetration of rings by linears leads then to a transient network. As both natural and synthetic polymers can be found in this severe cyclic architecture the present results are of both fundamental and practical significance. Optimized microprocessing as well as rheology modifications are thus within reach. Rings will be investigated further using adequately isotope-labeled systems to study differences in segmental dynamics and chain fluctuations compared to their linear analogs. Also the question whether a plateau modulus shows up or power law relaxation persists if the rings get considerably larger will be investigated. Additionally blends with linear homopolymers will be addressed as well to be compared with recent advances in blends of linear with dendritic polymers. The techniques of interest are neutron spin echo (NSE) and small angle scattering (SANS) in quenched state which focus on the short times i.e. high-temperature motion and long times i.e. large-scale dynamics respectively. The present work was carried out in the framework of the Joint Programme of Activities of the SoftComp Network of Excellence (contract number NMP3-VT-2004-502235) granted under the FP6 by the European Commission. FIG. 2: SANS experiments on dilute solutions under θ and good solvent conditions show the characteristic scattering vector dependences. q−2 is random-walk statistics on intermediate length scales whereas the q−1.6 is prominent for excluded-volume interactions in swollen rings. The strong parasitic forward scattering at low q for the cyclohexane sample following q−3.1 is due to density fluctuations in the theta-state. [1] M. Kapnistos, M. Lang, D. Vlassopoulos, W. Pyckhout-Hintzen, D. Richter, D. Cho, T. Chang, M. Rubinstein, Nature Materials, 7, 997-1002(2008) [2] M. Rubinstein, R. Colby, Polymer Physics, (Oxford University Press, 2003) [3] M. Rubinstein, Phys. Rev. Lett., 24, 3023-3026(1986) FIG. 3: Linear chains are percolating through bridging by the rings at low concentrations. The effect of the linear chain on the dynamics of a ring could be quantified by deliberately mixing in the sample again known amounts of linear chains and remeasure the relaxation modulus. It shows that even at concentrations of 1/50th of the overlap concentration of the rings, the relaxation time spectrum gets a new long-lived component which grows substantially with concentration. The viscosity rises and a plateau shows up again. The understanding of this is important to relate former experiments in literature on rings which were not purified so extensively as here and therefore always showed a plateau modulus value. 33 The pronounced minimum for the Φ=0. Details of this work can be found in [3]. the large difference in size of the mineral and protein allows a separate analysis of both constituents. were observed. However.5 hours after initiation the mineralization. M. 10 10 10 10 2 2. Pipich1. the role of these proteins in mineralization is not well understood. namely the crystalline polymorphs vaterite and aragonite. During the first 1. e. 1: Scattering pattern after 13 h mineralization in the presence of ovalbumin. S. So. This is a relevant goal as the capability of mineral formation by living organism is not yet well understood. Figure 2 shows the time evolution of the amplitude P4 of four relevant contrasts. the mineralization started with the formation of an amorphous polymorph. The formation and dissolution of 2+ amorphous CaCO3 is accompanied by Ca mediated unfolding and cross linking of about 50 protein monomers showing linear chain characteristic scattering. The Q-4 power law is a measure of the total mineral surface (Porod’s law: P4·Q-4) whereas the Q-1 power law is caused from the linear chains of ovalbumin. W. in order to identify the mineral polymorphs.Balz2. The symbol Δρ = ( ρ − ρ S ) describes the difference of the coherent scattering length density ρ of the mineral or protein and of the solvent ρS.Nucleation and Growth of CaCO3 Mediated by the Protein Ovalbumin V.2]. respectively. In all cases a peak becomes visible about 2.5 h the scattering of the protein is best described by the 34 . which after reaching a maximum volume fraction transformed to a crystalline polymorph. Small-angle neutron scattering (SANS) is a new technique in biomineralization which allows identification and structural analysis of biomineral composites by the method of contrast variation [2]. such as skeletal support or protection of soft tissues [1.05 (Table). This scattering was identified from ACC particles as the intercept of P4 = 0 occurred at Φ=0. With the particular intention of a better insight into the early stages of mineralization. D. Δρ = 0 . we performed studies of in-situ calcium carbonate formation mediated by the egg-white protein ovalbumin. such as proteins and polysaccharides.g.E. Living organisms are capable of developing inorganic minerals with complex architectures to fulfill important biological functions. A characteristic result after 13 h mineralization by using the gas diffusion technique is shown in Figure 1. The mineral phase of such materials is intimately associated with organic macromolecules. Starting from the amorphous phase several stages of mineralization. The protein complexes act as nucleation centers because of their enrichment by Ca2+ ions.55±0. Johann Gutenberg-University. Another important property of SANS is the large variation 2 range of scattering contrast Δρ of aqueous solutions by the exchange of H2O and D2O. The scattering from the mineral and protein are distinct by the two power laws We performed several mineralization experiments in aqueous solution of different D2O content. So. The corresponding values are summarized in the Table together with Φ representing the D2O content when the protein and the mineral polymorphs are matched in water. A particular goal was to achieve a better insight into the early stages of mineralization.82 sample after 5 h provide the interpretation that CaCO3 follows a polymorph sequence of amorphous → vaterite → aragonite (Table). The scattering patterns in Figure 3 represent the contribution from the protein during mineralization (large Q part in Figure 1). Tremel2. The scattering pattern is characterized by two power laws representing μm large mineral particles and nm-sized proteins at small and large scattering vector Q.74 D2O solvent after 4 h. Schwahn3 1 2 JCNS: Jülich Centre for Neutron Science Institute for Inorganic Chemistry. Wolf2. and the smallest scattering of the Φ=0. Mainz 3 IFF-5: Neutron Scattering dΣ/dΩ [cm ] Formation of CaCO3 mineralization in the presence of the egg-white protein ovalbumin was studied by time-resolved small-angle neutron scattering.5mg/ml Ovalbumin 1 Q -4 -1 0 Q -1 -1 10 -3 10 -2 -1 10 -1 Q [Å ] FIG. 10 4.05 0..74 0. The observed formation from less dense to more dense polymorphs follows the Ostwald-Volmer rule [1]. 3: Protein scattering during mineralization (Β before start.55 0. [2] [3] [4] 35 .49 3.25 0. In a separate experiment of ovalbumin in 0. Pipich et al.00 0 1 2 3 -2 10 -3 10 -2 10 -1 -10 74% 82% 4 -2 5 -1 6 7 8 Q [10 Å ] FIG. and water. Calcium carbonate nanocrystals are stabilized by surface bound ovalbumin which is eventually occluded between the individual CaCO3 crystallites.: SANS parameters of the calcium carbonate polymorphs. After 90 minutes a gradual transition to a Q-1 power law is observed which was complete after nearly 3 h (see also Figure 1).20 dΣ/dΩ [cm ] -1 Q -1 10 10 10 10 1 Q -2 0 Q -1 P4 [10 cm Å ] -4 20 10 0 5 4 3 2 1 0 0 ACC Vaterite -1 -1 0. i. ovalbumin. Heiss et al.81 0. V. ρ [10 cm ] Calcite Aragonite Vaterite Amorphous Ovalbumin Water 4.41 --- seen from the reduction of the amplitude P4 in Figure 2. (JACS) 130 (2008) 6879-6892. The enlarged scattering is caused by cross linking of about 52 proteins to an object with the conformation of a Gaussian linear chain -2 -1 as seen by the Q and Q power laws (Radius of gyration ≅600Å).30 0. Chem. 90 min. compact structure of the native state.64+1. we give a first clue concerning the role of protein mediated CaCO3 mineralization.76 0. the amorphous phase started to dissolve as [1] S. A peak from the amorphous polymorph (ACC) became visible at ≈ 3h.69 5. Schwahn et al.10 0.61Φ -0.50 40 30 H2O D2O 0. Chem. After 4h the minimum of the 0. Soc.6% increase in the protein scattering length density which can be interpreted as a “loading” of the protein with about 280 Ca2+ ions.1 M CaCl2 aqueous D2O solution.1 M CaCl2 aqueous solution we determined a cross linking of about 50 proteins (inset in Figure 3). J. A gradual aggregation of the proteins is observed. Oxford 2001. J. A.95Φ 10 -2 Φ 0. D. The inset shows ovalbumin in 0. TAB. The slight increase of scattering after 90 minutes is the result of a 14. Biointerphases 2 (2007) 16-20. after start of mineralization at 7 15 min . Mann. In summary. The strong increase in intensity at low Q indicated that the process was accompanied by a crosslinking ("salting out") of the protein molecules.74 D2O solution identifies the vaterite polymorph whereas aragonite appears as the stable phase within 12 h.29 1. After the protein was “salted out”.15 0. Am. thereby forming a mesocrystal. Ovalbumin first acts as a “cation sponge” which 2+ locally increases the Ca concentration. 2: Porod amplitude versus mineralization for different scattering contrasts. Phys. C111 (2007) 3224-3227. This scattering was interpreted in terms of a Gaussian linear chain with relatively large statistical segments. the structural change of the protein was a continuous process during this time interval.. Biomineralization. and ξ nearly 13h). In this time interval the formation of the amorphous polymorph proceeded and approached its maximum (Figure 2). These Carich pockets of ovalbumin aggregates seem to build nucleation centers for the incipient calcium carbonate formation. Oxford University Press. 2 4 6 8 10 12 Time [h] FIG.. an inorganic-organic hybrid material [4].e.561+6. Lund2. P.25% block copolymer in a solvent mixture with 90 mole% DMF.5 ms 194. Colmenero2 . This phenomenon was exploited by rapidly mixing a DMF solution with unimers with water/DMF pure solvent mixture by means of a stopped flow apparatus. 100 90 80 70 60 50 40 30 20 10 0 dΣ/dΩ/φ (Q) [cm ] 14. The stopped flow set-up was coupled to the small angle x-ray scattering (SAXS) instrument at the high brilliance beamline.Kinetics of Micelle Formation L. 1: Normalized absolute scattering cross sections at different times during the kinetics for the PEP-PEO system at a final polymer concentration of 0. This block copolymer forms starlike micelles in water and water/dimethylformamide(DMF)mixtures which are both selective solvents for PEO [3]. Using a detailed quantitative model we further demonstrate that the kinetic pathway proceeds by unimer exchange where only single chains are added or removed at a time. with respect to PEPallows an effective tuning of the micellization properties by varying the solvent composition. The route by which amphiphilic molecules selfassemble into nano-scale objects such as micelles is still not fully understood. Richter1 1 2 IFF-5: Neutron Scattering Donostia International Physics Center (DIPC). Donostia . D.02 0. The morphology of such systems has been widely studied during the past.03 The induced self-assembly process causes a strong increase in intensity directly reflecting the growth of the micelles in real time. A large difference in interfacial tension.25%. T. As a model system we employed a well-defined poly(ethylene-alt-propylene -poly(ethylene oxide) (PEP1-PEO20. Pmean. 36 . a detailed understanding of the mechanism and kinetic pathways of the selfassembly process has not been reached to date. Panine3.France. ID02. numbers indicate the approximate molecular weight in Kg/mole)block copolymer. The growth of the micelles in terms of an increasing Pmean is shown in Figure 2 on logarithmic time scale for three different concentrations.5 ms 2914. Grenoble. The solid lines display fit results from a standard core/shell model. In addition there is a prominent lack of detailed physical modelling of the data.5 ms 994.5 ms 240 s Unimer Reservoir Model fits -1 0. γ. results remain largely inconclusive. Narayanan3.San Sebastian. An important parameter which can be extracted by the model fit is the mean aggregation number. A typical example of the time evolution of the scattering curves is presented in Figure 1. We chose an optimized acquisition time of 20 ms. J. Willner1. at the European Synchrotron Radiation Facility (ESRF) allowing a synchronization of extremely fast mixing (4. Using a quantitative model we showed that the self-assembly process can be viewed as a primary micellization and growth process where the elementary growth mechanism is the exchange of single unimers A classical example and a model system for selfassembly are amphiphilic diblock copolymers that undergo micellization in aqueous solution [1]. Thus so far.5 ms 24. The data were obtained from a solution containing a total volume fraction of 0. By means of synchrotron x-ray scattering with millisecond time resolution we got direct structural information in-situ on the birth and growth of block copolymer micelles. -1 0. The formation of block copolymer micelles in selective solvents occurs spontaneously usually in the subsecond range. In this work [2] we show that the required nanoscale spatial resolution and millisecond temporal resolution could be achieved for an in situ investigation by using synchrotron x-ray scattering. M. The scattering data were analysed using a standard core shell model describing the detailed structural features of both the inner PEP core and the outer PEO corona of starlike micelles.01 Q [Å ] Fig. Monkenbusch1.5ms) with rapid data acquisition. In pure DMF only single chains (unimers) are present but as soon as some water is added the block copolymers spontaneously aggregate into micelles. Spain 3 European Synchrotron Radiation Facility. This is primarily due to the lack of experimental techniques having the correct spatial and temporal resolution with the combination of a suitable welldefined model system. R. The solid lines in Figure 1 represent fit results of the core/shell model. The excellent agreement indicates that at all times a starlike structure is adopted. The self assembly process of a model amphiphilic block copolymer system was triggered by an interfacial tension jump experiment by rapidly changing the solvent quality for one of the blocks. ). Consequently. the experimental data are sufficiently explained by insertion/expulsion of a single chain at a time. E. The whole micellar evolution can then be calculated by solving a system of differential equations which was done numerically by using standard routines and by fitting to the experimental data. extracted from the fits for three polymer volume fractionson a logarithmical time scale: 0.. R. I. disassemble to provide unimers. This process becomes exhausted at intermediate times leading to a “shoulder” of Pmean that changes with concentration. A further growth of the micelles requires that some of them. between PEP and water/DMF was found to be 19mNm compared to 12mNm obtained by pendant drop tensiometry.φ1) (φ1=unimer volume fraction).φ1). submitted to Phys. et al. jP+1: In summary. particularly the smaller ones. Following Neu et al. Applying a quantitative model. J.5% (triangles). Schmelzer ed. Phys. M P +U k+ M P +1 k- with MP as the number of micelles of size P and U the number of unimers. Pmean.macroscopically determined quantities. Macromolecules. the kinetics of formation of block copolymer micelles have been directly observed in situ by synchrotron small angle X-ray scattering with millisecond time resolution. Oxford . [2] Lund. The initial free unimers are rapidly consumed in a primary micellization event as shown in region leading to classical overnucleation. and 0. R.4566 (2006).C. et al. and flux.W. The Physics of Block Copolymers. k+/k. The contribution of other more complicated mechanisms.g. 061406 (2002).which in our case was taken as the difference of the free energy of a starlike micelle and an equivalent amount of unimers taking properly into account the translational entropy. like fusion and fission.. The kinetic pathway from unimers to the final micelle is schematically depicted in Figure 3. φ1 ))] which is only determined by the insertion rate constant k+ and the potential G(P. Oxford University Press. the interfacial tension. 39.P. we see that the formation and growth of micelles can accurately be described by a primary micellization and growth process governed by single unimer exchange mechanism. KGaA.25% (squares). γ. φ1 ) − G(P. et al. I II fast slow III Qualitatively the evolution of Pmean can be summarized as follows: At shortest times the data suggest the existence of a fast initial aggregation (t<≈5ms) that cannot be entirely resolved experimentally. 66. 2: Time dependence of the aggregation number. [4] «Nucleation Theory and Applications» (J. [1] Hamley . In order to quantitatively discuss the experimental data we have derived a kinetic model that involves simple unimer exchange as the single elementary growth step: Figure 3: Schematic view of the kinetic pathway in the formation of polymeric micelles. [5] Neu. All concentrations could almost perfectly be described by a consistent set of parameters. UK (1998).denote the insertion and expulsion rate constants which both depend on P. cannot entirely be excluded. however. which gives a net creation rate in terms of the concentration. The terminal relaxation towards a common equilibrium slows down with time.5] the formation and growth of the polymeric micelles is governed by the micellization potential. [5] we assume the validity of the “detailed balance” or “microscopic reversibility” principle. φP+1. Fig. jP +1 = k+ ( P)φ1[φP − φP+1 exp(G(P + 1. Within the context of classical nucleation and growth theories [4. At later times in region III the equilibrium is approached with a narrow size distribution of the final micellar entity. G(P.125% (stars). The parameters nicely compare with 37 . Rev. Lett. Weinheim (2005). e. metastable micelles with a broader size distribution are obtained in region II corresponding to the shoulder in Pmean at intermediate times in Figure 2. Rev. [3] Lund. The overall rate increases with concentration. Wiley-VCH Verlag GmbH & Co. 0. Solid lines represent a fit using the kinetical model described in the text. W. Fit results are shown as solid lines in Figure 2. the maximum displacement of a PEO segment observed during about 1 ns amounts to less than 1 nm. The system PEO / PMMA is in particular interesting because of the significantly different glass transition P EO P MMA temperatures (Tg ≈ 200 K. One important consequence of the fact. Julich.3 . So far we may state that the dynamics of PEO in a matrix of PMMA at temperatures where PMMA is nearly frozen is in good agreement with Rouse dynamics characterized by an average friciton. D. is at present matter of controversial discussion. In such a situation. PEO moves in the random environment created by the frozen PMMA component [2]. They show a temperature dependence very similar to the one found in pure PEO. The relevant parameter in this model is the friction of the observed chain in a heat bath representing the adjacent chains. One of the important questions is how the segmental friction arises if a segment is surrounded by a chemically heterogeneous environment. Genix2 . Here. 1: Mean square displacements obtained from backscattering spectra from the 35% PEO/PMMA sample at different Q and temperatures. 2 displays neutron-spin-echo (NSE) measurements of the single chain dynamic structure factor of PEO chains in the PMMA matrix measured at the IN15 (ILL. M. 20080 San Sebastian. Colmenero2. Tg ≈ 400 K). Spain 4 ¨ Halle-Wittenberg. Samples with different concentrations (25%. Richter1 . We present a neutron scattering study on the miscible polymer blend poly(ethylene oxide)/ poly(methyl methacrylate) (PEO/PMMA) which due to the very different time scales of motion is a perfect candidate to address this topic. This has been realized by preparing a mixture of protonated PEO chains in deuterated PMMA. 35% and 50% of PEO) have been measured at different temperatures between 350 K and 400 K. Monkenbusch1. that the segments of a chain are linked to each other (chain-connectivity). Grenoble) up to about 80 ns. The data follow nicely straight lines when plotted against the square root of time in good agreement with the Rouse prediction. E. The related values for the friction can be obtained from the slope of these lines. Arbe3 . ment (msd) of the PEO segments. ¨ Germany Donostia International Physics Center. By neutron backscattering spectroscopy we have followed the mean square displace- FIG.Polymer Chain Dynamics in a Random Environment K. Only exploring different time and length scales allowed to identify the mechanisms of dynamic heterogeneity on a molecular scale. Paseo Manuel de Lardizabal 4. however the friction is about one order of magnitude larger than in the pure system. Fachbereich Physik. Starry-eyed. Institute of Solid State Research . In order to achieve this result a deuterated PMMA matrix was used. one could assume that in a perfectly miscible polymer blend the friction of a given segment is determined by the average friction of the blend components. A. Straube4 1 2 FZJ. but very different in their dynamical properties on the other hand. A. A.Neutron Scattering.-C. Spain 3 Centro de F´ ısica de Materiales CSIC-UPV/EHU. Fig. and therefore that the above mentioned naive assumption is true. the relaxation of thermally activated fluctuations is balanced by entropic and viscous forces (friction). At present the investigation of dynamic miscibility in a blend of two different polymers is an active area of research. for temperatures around and beP MMA low Tg . D-06099 Halle. Germany Martin-Luther-Universitat The question of what determines the behaviour of two polymers which are perfectly miscible on the one hand. where a mixture of deuterated and protonated PEO was immersed. The dotted lines in Fig. 1 shows the result for a sample with 35% protonated PEO in 65% deuterated PMMA at two different momentum transfers Q. Apartado 1072. Strauch4 . 20018 San Sebastian. Fig. the experiments were performed at the instrument BSS at the FZJ in Julich. M. is that segments exhibit a mean squared displacement proportional to the square root of time different to the displacement for a Fickian diffusion which is increasing linearly in time. J. Depending on temperature. From this one could conclude that the effect of blending is not more than imposing a different friction to a given segment. Niedzwiedz1. We have investigated the PEO dynamics in PMMA by quasielastic neutron scattering. 2 are 38 . ¨ The dynamics of a polymer chain in the melt is well described by the Rouse model [1]. Wischnewski1 . Compared to the spectrum from pure PEO. quasielastic neutron scattering observes local processes. Y. Phys. For the blend system also backscattering data are included. 3. FIG. For the blend also the backscattering data are included. D. Colmenero. Lutz. Q-values are 1 nm−1 . Cao. Richter. The solid lines show the results of the model with randomly distributed friction.a result of a Rouse description based on the average Rouse friction obtained from the backscattering data in fig. Jones. Finally. the highest Q data are reproduced at very short times only. With this we obtain the solid line in Fig. From the decay we realize that a shift of the characteristic time from 1 ps in pure PEO to about 6 ps for PEO/PMMA is found –in excellent agreement with NMR data [3] and also with the ratio of the corresponding Rouse rates as determined by the backscattering experiment. 1272 (1953) [2] A. The solid lines in Fig. Rouse. J. 1724 (2003) 39 . 1. Ediger. L. Phys. the backscattering data. 2 show the best result which could be achieved for the 35 % PEO/PMMA sample. In the following we demonstrate that the contradictory experimental findings can be explained assuming that the mobile PEO chain experiences a heterogeneous environment imposed by the rather stiff PMMA matrix. the question remains where the extremely broad distribution comes from. J. However. Lin and A. We may roughly quantify this slowing down in fitting an effective smaller Rouse rate. M. A.F. Starting from the pure PEO spectrum we shift the relaxation rate by a factor obtained from the backscattering data for the blend. R. fig. Willner and D. Alvarez. Obviously this description fails grossly predicting a by far too fast decay. 3 in perfect agreement with the observed spectrum. The values of the width obtained by applying the same procedure to the other compositions and temperatures show the expected behaviour: the width of the distribution function increases with increasing PMMA concentration (in the limit of zero PMMA concentration it should go to zero) and within one blend it increases with decreasing temperature. In a second step we convolve the thus shifted PEO spectra with the log-normal distribution function using the result from the NSE data. The three lowest Q values are in very good agreement with the experimental data. This picture can be rationalized by a chain where the beads undergo a random friction with a distribution of friction coefficients. Genix. we now can try to describe the TOF spectra of the blend displayed in Fig. G. however.5).-C. 3: Fourier transformed TOF data of pure PEO and 35% PEO/PMMA at T = 400 K. Villigen) at T=400 K. The origin of this significant deviation at the highest Q and longer times is still unclear. 1. 2: Single chain dynamic structure factor of the 35% PEO/PMMA system at T=400 K. This reveals retardations by factors 4 to 20 (depending on PEO-content and temperature) compared to the 1 ns scale and gives clear evidence for an additional effect of blending which has not been seen in the BSS data. Arbe. Rev. FIG. the only adjustable parameter being its width. the blend data are strongly broadened demonstrating a broad distribution of local relaxation processes in the PMMA environment. H. A. 2 nm−1 and 3 nm−1 from top to bottom. Line through the blend data: see text. 031808 (2005) [3] T. though it might have to be related to additional confinement effects imposed by the matrix. The dynamic structure factor for such a chain has been calculated by starting with the Langevin equation for a polymer chain and introducing a set of bead mobilities following a log-normal distribution centered around the average mobility obtained from [1] P. Having in mind that at high Q we observe directly the distribution of relaxation times. The solid line through the PEO data describes a fit with a stretched exponential (β = 0. E 72.5 nm−1 . The dotted lines illustrate the Rouse theory with Rouse relaxation rate obtained from the backscattering data. The dashed base represents the elastic contribution to the scattering. Chem. Apparently the motion is strongly slowed down towards longer times or larger length scales. the width from the NSE-data and the shift of the relaxation rate from the BSS data. 21.3 shows Fourier transformed time-of-flight (TOF) data on the same blend measured at the FOCUS instrument (PSI. E. this agreement which combines data over 5 orders of magnitude in time is remarkable. He. Macromolecules 36. Having in mind that all parameters have been obtained from other measurements. At high Q. Some of the best instruments from the closed DIDO reactor were transferred to the new national neutron source FRM II in Garching close to Munich (Fig. and J-NSE have been in operation for users since autumn 2007 now. The second SANS KWS-1 and the focusing SANS KWS-3 will become available for users soon. Transferred instruments included the powerful small angle neutron scattering units KWS-1 and KWS-2. FIG. which comprised of up to 13 instruments.65 µeV with a signal to noise ratio of better as 600:1.JCNS Instrumentation T. This instrument is designed to serve the need of the solid state chemistry and geoscience communities. POWTEX is realised and financed in the frame of the BMBF "Verbundforschung" and build in a collaboration of RWTH Aachen University and the Forschungszentrum Jülich within the Jülich Aachen Research Alliance JARA with the Göttingen University as further partner. replacing the former SV-29 of FRJ-2. The new thermal time-of-flight spectrometer TOPAS. Access is organized by the JCNS User Office. Gutberlet 1 1 JCNS: Jülich Centre for Neutron Science On May 2nd . In addition to the FRM II location. 2006. A brand-new high intensity magnetism reflectometer MARIA is in construction to be operational in 2009. 1: View of neutron guide hall of FRM II. Regular operation and access or external users started in March 2008. a rigorous upgrade and renewal programme was embarked. Both instruments are expected to be operational in 2011. In addition of developing. ILL and SNS JCNS operates state-of-the art neutron instruments under a common scientific objective. KWS-3 has been rebuild and up-dated including a brand new mirror coating and will be available for users till end of 2009. At current SPHERES offers an energy resolution of 0. Europe and across the globe for the benefit of their scientific research goal. 40 . HADAS was transferred to FRM II to serve as test and development instrument TREFF. the diffuse polarized neutron spectrometer DNS and the reflectometer HADAS. 2006 the FRJ-2 research reactor at Forschungszentrum Jülich GmbH was shut down ending a successful scientific work of more than four decades. Modern chemistry and biology labs can be used for sample characterization and sample preparation. a diffractometer for biological structure determination BIODIFF is being built till 2010. and the new time-of-flight powder and texture diffractometer POWTEX will be installed at the new guide hall east of FRM II. JCNS operates outstations at the first MW spallation source SNS in Oak The JCNS reflects the unique role of Forschungszentrum Jülich GmbH within the German neutron scattering landscape based on a compelling in-house condensed matter research program focussing on neutron scattering in the areas soft condensed and biological matter and nanomagnetism and highly correlated electron systems. In addition to the relocated instruments the second generation backscattering spectrometer with phase space transformer SPHERES was built at FRM II in the frame of the "Verbundforschung" funding of the BMBF. the novel focussing VSANS machine KWS-3. KWS-2. Instruments operated by JCNS are open to scientists from Germany. Besides the necessary adaptations of the instruments. DNS. At the national and international leading sources FRM II. A new branch lab with 32 scientists and technicians has been established at the new research reactor FRM II in Garching since 2006. 1). The end of this research facility with neutrons marked at the same time the start of a new era for research with neutrons at Forschungszentrum Jülich GmbH with the timely establishment of the Jülich Centre for Neutron Sciences (JCNS) on February 16th . constructing and running high class neutron instruments at FRM II JCNS also offers users specialized and unique sample environment and laboratory facilities on-side. JCNS is further constructing four more brand-new instruments at FRM II: together with the Technical University of Munich. the Jülich neutron spin echo spectrometer J-NSE. 3: Number of proposals submitted in the year 2007 and 2008 on the neutron scattering instruments of JCNS. 2).5 Å) < τ < 350 ns (16 Å) high resolution GISANS polarisation analysis high intensity 10−4 < q < 10−3 Å−1 high intensity polarisation analysis middle resolution texture diffractometer energy resolution 0. JCNS gratefully acknowledges complementary funding support from the EU within the 6th Framework Programme through the Key Action: Strengthening the European Research Area.jcns.info. 41 . This engagement also provides German users additional access to the thermal triple axis spectrometer IN22 and the lifting counter diffractometer D23. Research Infrastructures in the project NMI3 . the erection of the ultra high resolution neutron spin echo spectrometer is nearing completion (Fig. In the framework of the ILL Millennium programme. USA and the highest flux neutron research reactor of the Institute Laue-Langevin ILL in Grenoble. 2/3 of the available beam time at JCNS instruments is distributed to external users by application for beam time reviewed by an international Scientific Review Committee twice a year. An overview of JCNS operated instruments is given in Table 1. IN12 will be relocated to a new guide end position and JCNS has committed itself to a complete overhaul of the instrument. which will lead to significant improved performance.16 to 1.Instrument BIODIF Facility FRM II DNS J-NSE FRM II FRM II KWS-1 FRM II FIG. Further information on the instruments of JCNS and Call for proposals for beam time can be found on the JCNS web pages at www. FIG. ILL and SNS JCNS provides external users unique access to world class instruments under standardized conditions at the neutron source best suited for their respective ex- KWS-2 KWS-3 MARIA POWTEX SPHERES FRM II FRM II FRM II FRM II FRM II TOPAS FRM II TREFF IN12 SNS-NSE FRM II ILL SNS Parameters cold neutrons protein crystallography cold neutrons polarisation analysis Fourier times 2 ps (4. JCNS instruments are open to scientists from Germany.84 Å−1 energy transfer up to 100 meV polarisation analysis polarisation analysis polarisation analysis Fourier time up to 1 µsec TAB. 2: Inside of the spin echo spectrometer at SNS. Within a CRG contract with ILL concerning the cold triple axis spectrometer IN12 is operated in a consortium together with the CEA Grenoble.65 µeV q = 0. Figure 3 shows the distribution of external proposals received in the four proposal rounds in 2007 and 2008 for the instruments of JCNS. 1: JCNS instruments. This next generation spin echo spectrometer will offer unprecedented resolution and dynamical range. This investment in the instrumentation of the SNS gives German users access to two further high class instruments: the backscattering spectrometer BASIS and the high resolution powder diffractometer POWGEN3.Integrated Infrastructure Initiative for Neutron Scattering and Muon Spectroscopy. Concerning the number of proposals the small-angle scattering machine KWS-2 clearly stands out. Europe and across the globe for the benefit of their scientific research goals. France. periments. With the various instruments at FRM II. At SNS. Ridge. 42 . like gellation and shearband formation. with specific surface properties. however.IFF-7: Soft Condensed Matter Director: Prof. dynamics and non-equilibrium phenomena on a microscopic basis with an open eye for possible technological applications. response of colloids to external electric fields. and the synthesis of new colloidal model particles. Due to the slow dynamics of colloids and the tuneable interactions between the colloidal particles. The aim is to understand structure. Jan K. dynamics of various types of colloidal systems in equilibrium. there are many transitions and non-equilibrium phenomena that do not occur in simple molecular systems. mass transport gradients. the equilibrium phase behaviour of mixtures of colloids and polymer-like systems. G. Dhont The Soft Condensed Matter group investigates the chemistry and physics of colloidal systems. Colloidal systems can be regarded as solutions of very large molecules which exhibit phase transitions and show non-equilibrium phenomena that are also found for simple molecular systems. induced by temperature ƒ ƒ ƒ dynamics and micro-structural properties of colloidal systems near walls and interfaces. phase separation kinetics and dynamics of suspensions of spherical and rod-like colloids under shear flow. The main topics that are studied include. interaction potentials and particle geometries. pattern formation. the effects of pressure on interactions. ƒ the phase behaviour. ƒ ƒ ƒ ƒ 43 . the location of phase transition lines and gellation transitions and the dynamics of colloids and polymers. Poland Mass transport of colloidal-like particles through networks is relevant for a number of separation-. This screening is quantified by the so-called hydrodynamic screening length κ−1 . For small tracer spheres. As far as we know.Tracer-Sphere Diffusion in Rod-Networks K.1a). In particular. Moreover. aside from three earlier papers of the present authors [4][6]. hydrodynamic interactions are important. Despite We use fd virus as a rod-like colloidal host particle. The majority of reported tracer-diffusion experiments of spherical particles in rod networks focus on proteins in suspensions of F-actin. depending on whether the tracer sphere is large or small in comparison to the mesh size of the network. In this case. There are fundamentally different mechanisms leading to slowing down of diffusion of a tracer sphere due to the presence of a rod network. where the sphere can move through the voids of the network without distorting the structure of the network (see Fig. purification. hydrodynamic interactions between the tracer sphere and the rods are screened. and does not exhibit polymerization/de-polymerization nor possible bundle formation (like F-actin. Kang 1 . Tracer diffusion can then be described with the neglect of hydrodynamic interactions [4]. the hydrodynamic screening length of isotropic and nematic rod-networks is determined from a newly developed theory and experimental tracer-diffusion data obtained with Fluorescence Correlation Spectroscopy (FCS). To gain in understanding on the physics that underlies the transport characteristics through such confining media. very long and thin rods. due to the very small van der Waals attractions between the fd-virus particles. Poznan. A. Patkowski 2 . The host networks in these references exhibit a quite complicated dynamics by themselves. Due to entanglement of the rods in the network. an attempt has been made in these papers to develop a microscopic theory for tracer diffusion of spheres through networks of stiff. J. which measures the penetration depth of shear waves into the network. the rod networks are stable over a wide range of salt concentrations. G. K. which offers the possibility to study diffusion in these networks as a function of the range of electrostatic interactions. there is no theory available yet for the hydrodynamic screening length. rather stiff rod (length 880 nm. have been reported : nucleosome core particles in dispersions of DNA [1]. For the rod networks considered here. which are relevant for mass transport in cells. Dhont 1 1 2 IFF-7: Soft Condensed Matter A. translational motion of the tracer sphere is only possible when the network structure is severely distorted (see Fig. Besides experimental work. Recently. wormlike micelles and 44 . Mickiewics University. FIG. there are no experimental data available on tracer diffusion of colloidal spheres in the much more simple quasi-static networks consisting of relatively stiff. thickness 7 nm and persistence length 2500 nm). colloidal spheres in solutions of "living polymers" [2] and colloidal spheres in dispersions of xanthan [3]. EHUT). which is indeed a very long and thin. 1: The two extreme cases where (a) the tracer sphere is large compared to the mesh size of the network. direct interactions between the sphere and the rods is much more pronounced than hydrodynamic interactions. where both (screened) hydrodynamic interactions and direct interactions between the tracer sphere and the rod network are explicitly accounted for. and (b) where the sphere is small compared to the mesh size. translational diffusion of tracer spheres in isotropic and nematic networks formed by long and thin colloidal rods is investigated as a function of ionic strength and rod concentration. For large tracer spheres.1b).and characterization techniques of macromolecular mixtures and might play a role in diffusive transport (of proteins) through crowded environments in cells. long and thin rods. experiments on tracer diffusion of spheres in host suspensions of slender particles. other than F-actin. Kang. but also by charge-charge interactions in case the rods and the sphere carry surface charges. as expected. The importance of the shadowing effect relative to hydrodynamic interactions can thus be varied systematically by changing the electrostatic screening length through variation of the ionic strength of the rod suspensions [6]. As can be seen. 0. the probability to find the sphere on the side-of-approach of the rod is larger than that on the "shadow side" of the rod. 124.[6]) and (b) 20 mM plus 100 mM added N aCl (from ref. Sacanna. The creation of mono-domain nematics through alignment in a magnetic field and the measurement of orientational order parameters of the nematic samples is discussed in that reference. This distortion is due to the fact that. [3] G.0 mM . A. The way to extract hydrodynamic screening lengths from the diffusion data. A.80 mM . J. J. (b) The screening length for the highest buffer concentration and 100 mM added salt in the isotropic and the nematic phase versus the fd-concentration. Buitenhuis. there is a strong effect of the ionic strength on the diffusive behaviour of apoferritin. 9 FIG. Bouteiller. J. Aarts.P. 45 .0 mM plus 100 mM N aCl). Phys. Here. A. Rädler.16 mM (from ref. is extensively discussed in ref. 214501 (2007). 044905 (2005). D.A. J. Biophys. Knoben.3b. L. Kang. and 20. 044907 (2006).M.3a. W. the hydrodynamic screening length increases with increasing fd-concentration in the nematic state.16 mM ♦. based on the newly developed theory. Buitenhuis. Chem. 2: Long-time self diffusion coefficients of apoferritin in fd-virus particle suspensions for two ionic strengths. Dhont. 3: (a) The hydrodynamic screening length κ−1 in units of the rod length L for the isotropic state versus the fdconcentration. in Fig. van der Gucht. A. there is nevertheless a distortion of the pair-correlation function between the sphere and the rods. J.H.K. Chem. Dhont. J. N. The vertical grey bars indicate the two-phase. 85.16 mM . Rev. The rod-sphere pair-correlation function is therefore distorted. Wilk. the distinction between directions perpendicular and parallel to the nematic director should be made. and decreases with increasing fd-concentration.[6]. Cohen Stuart.P.A. The screening length κ−1 for various ionic strengths scale onto a single master curve. Meier. The hydrodynamic screening length as a function of the fd-concentration for isotropic networks is shown [1] S. M. when the sphere moves past a rod. Rev.00 mM . Phys.G. In Fig. Koenderink. Tracer diffusion constants of apoferritin in fd-virus suspensions were measured with Fluorescence Correlation Spectroscopy (FCS). [4] K. Wilk. Besseling. A. G. 126. M. Philipse. S.2 the measured diffusion coefficients are plotted as a function of fd-concentration for two analytical TRIS/HCl-buffer concentrations that are used (0. J. giving rise to a force on the sphere that affects its diffusive motion. [5] K. J. 1817 (2003). Kang. Gapinski. E 67. Patkowski. (a) TRIS-buffer concentration of 0. [2] J. Chem. The shadowing contribution is not only determined by hard-core interactions. M. Keller. Phys. A. Lettinga.K.G. 4. The hydrodynamic screening lengths for the nematic phase are given in Fig. Patkowski. E 69 (2004) 021804. [6] K.G.G. the resulting contribution to the correlation function must be subtracted [5]. J. S. Phys. Since fd-virus particles fluoresce to some extent.[5] as "the shadowing effect". Surprisingly. 20 mM + 100 mM N aCl . This is confirmed from measurements of κ−1 at a fixed fd-concentration but different orientational order parameter through the variation of ionic strength [6]. Dhont. J. 051106 (2003).the fact that the network structure remains essentially unchanged during the diffusive motion of such a small sphere. Phys.A. J. isotropic-nematic coexistence region. This is probably due to the increasing degree of alignment of the rods with increasing concentration. The symbols refer to the different buffer concentrations : cT = 0. It thus seems that the hydrodynamic properties of nematic networks is highly sensitive on the orientational degree of order. 20. 122.[5]). FIG. We referred to the distortion of the pair-correlation function in ref. Mangenot. Ratajczyk. Patkowski.K. fd-virus. which are rodlike mono-disperse particles and nonadsorbing neither on glass nor on polystyrene latex particles. < D >.075 g/L. Aqueous buffer solutions (20 mM TRIS) of PS latex spheres with a radius of R = 85 nm were investigated with two different fd–virus concentrations. that is. In the first order approximation. To our surprise. and they were diluted from their stock solutions to a volume fraction of 2 × 10−4 . So far. Further. is less pronounced except in the immediate vicinity of the wall [1]. They have a contour length of 880 nm which is comparable to the maximum penetration depth applicable in an EWDLS experiment. which keeps the data treatment on a tractable level. We note that the Derjaguin approximation is not valid at this radius/rod length ratio. because the strength of the depletion potential decreases with decreasing R/L at constant density. From this. at this rod concentration.Enhanced Slowing Down of the Colloidal Near Wall Dynamics in a Suspension of Rods P. we will show the influence of rodlike particles. the depletion potential mediated by fd–virus is expected to be effective throughout the entire scattering volume.05 and 0. Further. The PS latex spheres are charge stabilized by sulfonate surface groups.5kB T . neither theoretical nor experimental. P.17 g/L. which can be introduced in the expressions for the initial relaxation rate Γ of the time auto correlation function of the scattered intensity. since the Debye screening length is in the range of 3 nm. the diffusion of the colloids is reduced by less than 20% [6]. but it will give a lower boundary for the required rod number density. above and below the overlap concentration. on the diffusion of spherical polystyrene colloids close to a wall. while the effect on the parallel diffusivity. there is a simple closed analytical form. we used the first order approximation to estimate the lowest rod concentration at which the depletion potential would cause a notable effect on the near wall dynamics of spherical colloids with a radius of R = 85 nm. the solvent viscosity will be changed only by a few percent and the fd–virus contribution to the scattering is essentially negligible. In other words. to third order in rod number density [5]. with which we can determine the parallel and normal component of the diffusivity 46 . R. To illustrate the effect of the fd-viruses on the colloidal diffusion close to the wall correlation functions g1 (t) are presented in figure 2 at a constant penetration depth of 2/κ = 270 nm and a total scattering FIG. Our experimental TIRM data for the depletion potential mediated by fdvirus is in quantitative agreement with these predictions. Holmqvist. independently [3]. The slowing down and the anisotropy of Brownian motion close to a wall due to hydrodynamic drag forces has been theoretically predicted [2] and recently experimentally verified [3]. Kleshchanok. the particles may be regarded as hard spheres. we found a much larger effect on the near wall particle dynamics than expected from these considerations. Therefore. is strongly affected by the presence of the rods. we performed EWDLS measurements with our tipple axis setup. we chose as a starting point a rod concentration of twice the overlap concentration which would give a contact potential of approximately 0. Lang IFF-7: Soft Condensed Matter In this report. 0. At these conditions. D. c∗ = 0. only the case of particles interacting by excluded volume with a wall has been considered. A schematic picture of the system close to the surface under evanescent illumination can be seen in figure 1. The sphere diffusivity normal to the wall. while the mean interparticle distance is of the order of several thousand nanometers. The depletion potential mediated by rodlike particles has been calculated by Mao et al. 1: Schematic picture of the sphere/rod system under evanescent illumination close to the surface We chose to apply fd–virus as a depletant. The effect of depletion on the dynamics close to a wall has received very little or no attention. < D⊥ >. In bulk solutions with the same fd–virus content. 16.. K. the slope of the g1 (t) at Q⊥ > Q is significantly smaller than that at Q⊥ < Q .. but the anisotropy increases. Phys. for Q = 0. P. M. Choi. P. while for the low fd-virus concentration the anisotropy is close to what can be expected for a system without depletant.. Kleshchanok. a significant reduction can be seen as compared to the nondepletant system also at large penetration depths (distances) for both fd–virus concentrations. However. 2: Initial decay ln(g1 (t)) for 2 × 10−4 volume fraction PS latex spheres (R = 85nm) in cf d = 0.0136 nm−1 (open triangles). Qtot = 0.⊥ > drop more strongly at low penetration depths.. [2] Brenner.. Exp. The anisotropy of the diffusion can nicely be seen as the separation between < D > (black squares) and < D⊥ > (red circles) for both concentrations.. 106. while a large difference is obvious for the EWDLS correlation functions. Chem.00785 nm−1 and Q⊥ = 0. Not only do the near wall dynamics slow down significantly.05 g/L (solid symbols) and cf d = 0. If one looks at the normal diffusivity. a pronounced effect can be seen for both fd–virus concentrations. for two different combinations of Q and Q⊥ together with the bulk g1 (t) for a fd–virus concentration of 0. Meier. It is notable that the anisotropy is always larger for the high fd–virus concentration. vector. In figure 3.0136 nm−1 (green).17 g/L at Qtot = 0..05 g/L (solid sym- bols) and 0.. 2/κR 5. G. If these data are compared to the expected g1 (t) for the same system without depletant [3] (lines in figure 2). R. G. Chem. This effect is more pronounced in the solution with the high fd–virus concentration. is determined for the two different fd–virus concentrations. [3] Holmqvist. G. Gapinski. 122. D. The lines are the expected ln(g1 (t)) for the same system without fd–viruses for bulk (black). Lang. J. Sci. In bulk. < D⊥ >. As for < D >.. Lekkerkerker. Lang. J. systematic measurements of the diffusivity parallel and normal to the surface at different penetration depths were performed. 242 (1961). Dhont. E. the procedure described above was performed for a series of six penetration depths.00785 nm−1 (open circles ). are plotted against the reduced penetration depth. we show the expected curves for the colloid solution without fd–virus as solid lines. R. To get a more complete picture of the effect of fd–virus on the colloidal dynamics close to the wall.17 g/L. 021402 (2006). < D⊥ >. Eng.0157 nm−1 and 2/κ = 270 nm for bulk (open squares) and for two different combinations of Q and Q⊥ as follows: Q = 0.. 2/κR.. and Q = 0. The lines are the expected curves for the colloid solution without depletant. are plotted against the reduced penetration depth. A.00785 nm−1 and Q⊥ = 0. and the normal diffusion. Phys. < D⊥ > decreases with decreasing penetration depth. M. [4] Kihm. A. K. Y. which indicates a strong anisotropy of the near wall diffusion. Lettinga. [1] Holmqvist P. Chem.. Ratajczyk. H. M.00785 nm−1 (red). D0 . E 74.. Using the same procedure described in our recent papers [3] the mean diffusivities for the parallel.17 g/L (open symbols). T. If one compares the data with the expected mean diffusivities without depletant (solid lines). no effect on < D > can be seen at large penetration depths for both fd–virus concentrations. Further. Tagaki. This slowing down of the diffusion close to the surface cannot be explained solely by the small reduction of the bulk diffusion. and for the high fd–virus concentration system < D . J. it is clear that the sphere near wall dynamics is affected much more than the bulk dynamics by the presence of the rods. 12010 (2007). K. [5] Mao. that is. and for Q = 0. For comparison. The EWDLS g1 (t)) show a much smaller slope than that of the bulk curve. FIG. < D > is decreasing much more with decreasing penetration depth than was observed for the system where no depletant was present.. Banerjee. the g1 (t) values with and without the fd–virus are almost identical (the 20% reduction is hardly measurable). the extracted mean diffusivities normalized to the bulk diffusion constant. Fluids 37. K.17 g/L (open symbols). W.FIG.0157 nm−1 . Patkowski. 2/κR (where R is the colloidal radius). Langmuir 23. Dhont. < D >. Rev. 044905 (2005). C. 3721 (1997). [6] Kang. To investigate this in detail. D. J.0136 nm−1 and Q⊥ = 0. H. 47 .. P. Phys. 811 (2004). D0 . for cf d = 0. P. N. K. Buitenhuis. 0. J.. J. Cates. 3: < D > (squares) and < D⊥ > (circles) normalized to the bulk diffusion.0136 nm−1 and Q⊥ = 0. at penetration depths below about 450 nm. . with a full account of the many-body HIs. A.Dynamics in colloidal suspensions: from neutral to charged particles G. The scope for these systems has been broadened even further through the increasing importance of biophysical research dealing with charged biomolecules such as proteins and DNA [1]. Argentina We have made a comprehensive study on the dynamics of suspensions of charge-stabilized and neutral colloidal spheres. 3]. In addition. The simulations have been performed. in particular. in the framework of the model of dressed spherical macroions interacting by an effective pair potential of screened Coulomb type [2. Lines: theoretical results. and the possibility of measuring self-diffusion in a scattering experiment at an experimentally accessible wavenumber. Numerous short-time dynamic properties including diffusion functions. Many of the theoretical and computer simulation methods developed in colloid physics are applicable as well to biological molecules and cells. the wavenumber-dependent diffusion function D(q ) determined in a scattering experiment. These interactions cause challenging problems in the theory and computer simulation of the colloid dynamics. charges and added salt concentrations have been considered. the so-called hydrodynamic function H (q ). Through comparison with the simulation data. (a) Charged particles in salt-free solvent (water). i. Banchio 2 1 2 IFF-7: Soft Condensed Matter Universidad Nacional de Córdoba. Nägele 1 . and the rotational and translational short-time self-diffusion coefficients Dr and Ds . we report on extensive Stokesian Dynamics (SD) simulations where the influence of electrosteric and hydrodynamic interactions has been studied for numerous short-time properties including the high-frequency viscosity η∞ . to the high-frequency shear viscosity η∞ . respectively.J. All depicted quantities are normalized by their zero concentration values marked by the subindex 0. spanning the range from hard-sphere systems to low-salinity suspensions where the charged particles repel each other over long distances. 1: tems with differing particle concentrations. The results of this study were used. FIG. D(qm ). A large variety of sys- Simulation test of short-time GSE relations relating the rotational and translational self-diffusion coefficients Ds and Dr . translational and rotational self-diffusion coefficients and the high-frequency viscosity have been computed by means of a powerful accelerated Stokesian Dynamics simulation method. In this communication. Symbols: SD simulation data.e. Charged colloidal particles interact with each other directly by means of a screened electrosteric repulsion. nanosized particles not much bigger than the solvent molecules. and through solvent-mediated hydrodynamic interactions (HIs). to explore the validity of generalized StokesEinstein relations. suitably modified methods from colloidal physics can also be applied to dispersions of very small. (b) Suspension of neutral colloidal spheres. the applicability and the level of accuracy of analytical methods 48 . surface chemistry and food science. and the cage diffusion coefficient. From [2]. The dynamics of suspensions of charged colloidal particles is of fundamental interest in soft matter science. respectively. H (q ) would be a constant equal to one. McPhie and G. Nägele. J. which are frequently applied in the description of the colloid dynamics. Indeed. For all systems examined in our SD study. Nägele. However. From [3]. micron-sized colloids down to proteins in the nanometer range such as lyzozyme and apoferritin. Phys. and hydrodynamic function H (q ) = S (q ) × D(q )/D0 of a salt-free suspension of charged particles. As shown in this figure.: Condens. The general trends in the concentration and salt-dependence of the transport coefficients predicted in our SD simulations are in agreement with experimental findings on chargestabilized suspensions and neutral particle systems. Ds ≈ D(qs ). Our simulations show further that rotational self-diffusion. in these systems there is no isobestic point for S (q ) and H (q ). The non-existence of a φ-independent isobestic point in salt-free suspensions is exemplified in Fig. Without HI. Matter 20. D(q ). As can be noticed. D(q ) and the hydrodynamic function H (q ) for a salt-free suspension of charged particles. 2. J. [3] A. Chem. 1a). FIG. 1b). independent of the colloid volume fraction φ. applies reasonably well to neutral hard spheres and to high-salinity systems (see Fig. DLS at the specific point qs can be used to obtain a decent estimate for the value of Ds . Thus. The assessment of the quality of these relations is a necessary prerequisite for the experimentalist interested in deducing rheological information from dynamic scattering experiments. 1 by a horizontal line of height equal to one.corresponding GSE relations. The function H (q ) is a direct measure of the HIs. Ds ≈ D(qs ) is obeyed indeed within reasonable accuracy. Banchio and G.J. from systems of large. This leads to the occurrence of an isobestic wavenumber qa ≈ 4. 2: and ad-hoc concepts has been tested.G. Consequently. In low-salinity suspensions. The dynamic quantities D(q ) and H (q ) are scaled by the respective short-time selfdiffusion coefficient Ds . E 78. M. 1 for an illustration of this important point where simulation results for the static structure factor S (q ) at various volume fractions are compared to corresponding results for D(q ) and H (q ). related to the position. 060401(R) (2008). independent of the volume fraction. qm . both for charged and neutral particles. The assumption made here is that the dynamic scattering function at such a wavenumber is essentially determined by selfdiffusion. 404213 (2008). and to a lesser extent also translational self-diffusion. Phys. Phys. of the static structure factor peak. the SD simulation scheme has been used to scrutinize the validity of short-time generalized Stokes-Einstein (GSE) relations that provide an approximate link between diffusion coefficients and the high-frequency viscosity. and the particle concentration. normalized short-time diffusion function. the GSE relation for the cage diffusion coefficient. our comprehensive SD simulation study of short-time transport properties forms a useful database for researchers interested in information on the suspension dynamics of globular colloidal particles. See Fig. where D(qs ) is the diffusion function measured at qs . 128. 2 includes SD simulation results for S (q ). It has been suggested that the (short-time) selfdiffusion coefficient can be probed in a dynamic light scattering (DLS) experiment at a specific wavenumber qs located to the right of the principal peak in S (q ). Colloidal hard spheres have a common static and hydrodynamic length scale set by the particle radius a. S (q ). GSE relations are fundamental to a growing number of microrheological experiments.02 where both S (q ) and D(q )/Ds are equal to one. SD results for the static structure factor. This finding is of relevance in numerous scattering experiments on colloidal systems where the large wavenumber regime is not accessible experimentally. In summary. without noticeable collective diffusion contributions. All dynamic quantities are normalized by the self-diffusion coefficient Ds . As an important application. where S (qs ) = 1. if this assumption is valid at least on an approximate level. Our simulation results show that the accuracy of a GSE relation is strongly dependent on the range of the interaction potential. 104903 (2008). it is strongly violated in the case of salt-free suspensions of charged particles (see Fig. A strictly valid GSE relation would be represented in Fig. and where alternative techniques to measure Ds directly are not applicable or unavailable. 49 . the geometric mean particle distance becomes another length scale of physical relevance.G. The dashed vertical lines mark the wavenumber qs where S (qs ) = 1. as a function of the wavenumber q times the particle radius a. D(qm ). Nägele. Fig. [2] A.J. Rev. A simulation analysis of the long-time dynamics of charged colloidal spheres with a full account of the HIs remains as a major challenge which we plan to address in future work. McPhie and G. 78. are faster than predicted by the [1] M. we find the difference between D(qs ) to be less than ten percent. Banchio. independent of the scattering wavenumber. But also surprisingly straight silica rods may be formed. (B) a rod with a clear coreshell structure. Depending on the conditions silica nanowires can be formed. In contrast to the complicated genetic engineering FIG. Their strategy is to modify the coat protein of M13 via genetic engineering specifically for each metal or oxide. Single silica rods with high uniformity. the filamentous fd virus is used as a template to regulate the formation of silica nanomaterials with well-defined morphologies. sensors and catalysis[2. Hammond and co-worker as a template in the synthesis of metallic and other magnetic and semiconducting nanowires[4]. etc. Buitenhuis. because most inorganic materials do not form the desired structure by themselves. an assembly of three rods is indicated by the arrow. (A) rods with a uniform silica layer and semi-spherical ends. 2) nanowires with a curved shape and 3) bow-tieshaped bundles with well-defined shape and hierarchy.Synthesis of silica rods. the diameter is constant and the silica layer is homogeneous. The surface of these rods is smooth under the maximum 50 . differing only in one amino acid per coating protein. rods. see fig. In this paper. Along the axis of the rods. However. Zhang IFF-7: Soft Condensed Matter We explored fd as a template to direct the formation of silica nanomaterials with different morphologies through simple sol-gel chemistry[1]. electronic and mechanical devices. 1: TEM images of typical rods. (C) a slightly curved rod where no core-shell structure is visible. route used with the M13 virus. The synthesis of these anisotropic nanostructures is a big challenge. biological and organic materials. usually have a well defined structure down to the nanoscale. and under other conditions bow-tie-shaped bundles of rods are formed. Z. which seem to accurately transcript the bending conformation and the length of the fd viruses in solution. M13. using acid-catalyzed hydrolyzation and condensation of tetraethoxysilane as silica precursor. aggregated silica-virus hybrid nanorods as well as pronounced granular silica are observed for sample type 1. (D) EDAX analysis of the rod shown in C. a virus which is almost identical to fd. three kinds of morphologies are observed: 1) silica nanorods with a diameter of 20 nm and a homogeneous silica layer. are in the focus of research interests due to their potential applications. for example in optical. fibers. The results described in the present paper may serve as a basis for the further development of the synthesis of inorganic materials using biopolymers as a template. wires. Fd viruses have a length of 880 nm and a diameter of 6. we show here that wild-type fd virus can also be used as a template in the synthesis of inorganic materials using simple sol-gel chemistry. As far as we know.6 nm. there is no report concerning the application of fd or M13 as a template for silica precipitation. especially supramolecular systems. we are the first to use fd as a template for material synthesis and have observed several interesting structures. One dimensional anisotropic inorganic nanostructures such as tubes. as far as we know. wires and bundles using filamentous fd virus as template J. 1. 3]. Using (bio)organic materials as a template to build up anisotropic inorganic nanostructures has therefore emerged as a highly attractive method in recent years. which have a remarkably well defined shape and dimension. so that the coat protein can selectively induce precipitation or assembly of that specific metal or oxide on the surface of the virus. Under different conditions. has been intensively explored by Belcher. In contrast to inorganic systems. the silica coating is highly uniform. Silica wires seem to transcript the bending conformation and length of intact semi-flexible fd. most of the rods are straight and only a few slightly curved rods are seen (fig. D. Y. 2). Chem. This morphology is remarkable because as far as we know. no similar morphology has been reported for any other virus or organic template in the past. These results imply that most fd remains intact during wire formation (an exception is the broken wire shown in the inset of fig. T. 2446. where many rods much shorter than fd are observed. Yin. However. [2] Y. R. Yang. here. S. comparable to the total length of two intact fd viruses joined with each other head-to-tail (see the cartoon in fig. Small 2007. The curved shape of these wires probably originates from the bending configurations of the semi-flexible fd virus in aqueous media. 213. 3) if the aqueous straight rod sample of type 1 is mixed with a methanol/ammonia mixture. G. Q. Yan. Y. The low contrast part might be fd. A possible subunit of the bundles. Mayers. 1c). R. A. curved wires are observed entangled with each other (sample type 2. wires and bow-tie-shaped bundles. Zhang. [4] C. [3] G. D. The longer wires seem to consist of two viruses sticking together by partial parallel overlap. some rods do not show such low contrast part and look like pure silica rods (fig. 41. semi-flexible fd. The maximum length along the y axis of the well-defined bundles is about 2000 nm. J. G. but the exact reason for the shape and size of the bundles remains unclear. but under somewhat different reaction conditions also remarkably straight silica rods are formed that have a high uniformity in terms of the thickness and homogeneity of the silica layer. 2 by the white arrow). Nesper. F. 353. given that no agents containing nitrogen or phosphorus were used during the synthesis. 2003. Georgiou. M. [1] Z. The formation of the bow-tieshaped bundles seems to originate from an aggregation of (silica coated) fd viruses (and granular silica) after addition of the methanol/ammonia mixture. Work devoted to further understanding the results obtained in this paper and exploring the above problems is ongoing. F. A.Int. in contrast to the case of the straight rods described above. The bundles all have similar dimensions. 3: TEM image of bow-tie-shaped bundles dispersed in the background of granular silica. Wu. The surface of these wires is less smooth than that of the straight rods described before. The "pure" silica rod shown in fig. R. B. while long wires with a length twice that of fd are also observed. B. long. which might form from the silica coating of the fragments of decomposed fd. 303. Therefore. 3). 1c was subjected to EDAX analysis. Sweeney. P. Y. We demonstrated the capability of fd viruses to be used as a template for the formation of 1D silica nanomaterials. in Advanced Materials. Gates. FIG. a silica fan is indicated by the arrow. 51 . Angew. 2: TEM of nanowires. D. Buitenhuis. Some of the rods show a clear core-shell structure with a low contrast part along the center of the whole rod (fig. and the diameter of these wires shows a less sharp distribution with an average diameter of about 23 nm. Mao. these hybrid silica wires show an example of a quite precise transcription of the template. 1d). Apart from silicon and oxygen. Solis. The contour length of these wires is in the range of that of intact fd. 1c). N. Edit. Also the diameters of different rods are all close to the average value of about 20 nm. B. Y. Although fd is semi-flexible and somewhat curved in dispersion. Y. 2002. 15. Y. Belcher. 424. Xia. B. J. resolution of the TEM we used and the shape of the ends of the rods is semi-spherical. Bow-tie-shaped bundles of silica rods are formed (fig. At somewhat different reaction conditions. Krumeich. From a single rod point of view. Patzke.FIG. Kottmann. Whether or not the silica coating solidifies the fd virus completely so that the flexibility is lost is not clear. 1b). nitrogen and phosphorus are detected (fig. Three nanostructures with distinct morphologies have been observed under different sol-gel conditions using TEOS as silica precursor: silica rods. Science 2004. Kim. large differences in length are seen for different rods. However. Hayhurst. A broken wire can be seen in the inset as indicated by the arrow. Iverson. D. Sun. Reiss. Inset: schematic drawings of possible structures of the bundles. Long rods with a length comparable to the length of intact fd are observed along with short rods. The nitrogen and phosphorus can only be attributed to fd. fig. B. 3. as well as smectic layers by differential interference contrast (DIC) microscopy. 1: (a) Overlay of DIC and fluorescence images.2) form several lyotropic liquid crystalline phases. Fig. The existence of a smectic phase in suspensions of hard rods is an evidence of the high monodispersity and therefore of the model system character of such filamentous viruses.6 nm. The diffusion rate corresponds with the rate calculated from the diffusion in the nematic state with a lamellar periodic ordering potential that is obtained experimentally. in a suspension of rod-like viruses. The self-diffusion in a nematic phase formed by rod-like viruses [2] represent a recent examples. in particular the chiral nematic (cholesteric) phase and the smectic phase. E. a diameter of 6.Self-diffusion of Rod-like Viruses Through Smectic Layers. France We report the direct visualization at the scale of single particles of mass transport between smectic layers. showing smectic and two fluorescently labeled particles. 115 Avenue Schweitzer. CNRS-Université Bordeaux 1. Lettinga 1 . Grelet 2 1 2 IFF-7: Soft Condensed Matter Centre de Recherche Paul Pascal. The cartoon shows the jump of rod-like particle between adjacent smectic layers. Suspensions of fd rods in aqueous solution (20 mM Tris. The system of rods used in this work consists of filamentous bacteriophages fd. overcoming an energy barrier known as the smectic order parameter. 1(a) shows a sequence of images of a single region where both techniques are combined. In this way we have directly observed permeation of single rods in adjacent layers. the rods need to jump between adjacent smectic layers.2 µm. A comparison of the images shows that some rods jump between two layers while others remain within a given layer. Since the pioneering work of Onsager on the entropy driven phase transition to a liquid crystalline state [1]. This process of interlayer diffusion. In experiments various methods have been applied to obtain the ensemble averaged self-diffusion coefficients. For parallel diffusion to take place. Here we use video fluorescence microscopy to monitor the dynamics of individual labeled colloidal rods in the background of a smectic mesophase formed by identical but unlabeled rods. In this mesophase the particle density is periodic in one dimension parallel to the long axis of the rods. and a persistence length of 2. M. 1(b) in the direction parallel (z) and perpendicular (x) to the director. which are semi-rigid polyelectrolytes with a contour length of 0. FIG. 33600 Pessac. pH 8. was first predicted by Helfrich [3]. or permeation. The layer spacing is L 0. and occurs by quasi-quantized steps of one rod length. while the interparticle correlations perpendicular to this axis are short-ranged (fluidlike order). Self-diffusion takes place preferentially in the direction normal to the smectic layers.9 µm. also called permeation. The green lines indicate the time for which one particle stays in a given layer. Only a few studies have been done where dynamical phenomena are probed at the scale of a single anisotropic particle. but has never been verified experimentally for lyotropic systems. The trajectory of one of the rods is plotted in Fig. This figure summarizes the key observation: the diffusion throughout the smectic layers takes place in quasi-quantized steps of one rod 52 .88 µm. Knowledge of the dynamics at the single particle level is fundamental for understanding the physics of mesophases with spatial order like the smectic (lamellar) phase of rod-like particles. (b) Displacement of a given particle in the direction parallel (red line) and perpendicular (black line) to the normal of the smectic layers. where the diffusion parallel (D ) and perpendicular (D⊥ ) to the average rod orientation (the director) has been measured. P. The colloidal scale of the fd bacteriophage facilitates the imaging of individual rods by fluorescence microscopy. the structure and the phase behavior of complex fluids containing anisotropic particles with hard core interactions has been a subject of considerable interest. Understanding of the particle mobility in the different liquid crystalline phases is more recent. [5] M. 1949. it shows that the diffusion within the smectic player is extremely slow. FIG. Since the diffusion within the layer is glasslike. but this ordering potential nor its height had never been directly observed. 1969. The diffusion is strongly anisotropic in the direction normal to the smectic layers and quasi-discontinuous due to the presence of the layers.. Ann. see Fig..46. This implies that the permeation is a function of position z . increases in the smectic phase. Phys. Rev. which is about 20 in the nematic phase [2]. This is exemplified by the self-van Hove function G(z.36 kB T . which is the probability for a displacement particle to a position z after a time t. 3. istic of a sub-diffusive behavior. while γ > 1 is referred to as super-diffusion. Grelet. Phys. The ordering strongly influences the diffusion of the particles. For parallel diffusion the cage is formed by the energy barrier imposed by the smectic layers. t) for a fluid made of Brownian particles. as shown in Fig. where the discrete peaks in the selfvan Hove function are observed. The time evolution of the MSD given by ∆r2 (t) ∼ tγ provides the diffusion exponent γ : γ < 1 is character- [1] L. Rev. Moreover. [2] M. The parallel motion is close to be diffusive (γ 1) in the nematic phase (γ = 0. 2. Indeed. giving an amplitude of U0 = 1. 3: (a) Evolution of the self Van Hove function at different times. Since single particles are experimentally identified. 23. The potential can be best fitted with a sinusoidal. [3] W. i. In contrast. Sci. The functions are normalised to one. [4] B.FIG. P. 2005. 35. 2007. G(z. Lettinga et al. The “hoppingtype” diffusion is the consequence of the underlying ordering potential imposed by the smectic layers. Lett. 3059. 2: The mean ordering potential in the z-direction obtained by applying the Boltzmann on the probability density function of the center of mass with respect to the average position within the layer. This anomalous subdiffusive behavior suggests that a “cage escape” mechanism is at hand for both parallel and perpendicular diffusion. Europhys. which is obtained directly from our measurements. Thus permeation can be described in terms of Brownian particles diffusing in a one-dimensional periodic potential. D /D⊥ . due to the ordering potential.81. 51. A smooth gaussian distribution that smears out over time is expected for G(z. 372. N.P. the z-axis is scaled by the smectic layer thickness L.95) and reduces in the smectic phase to γ = 0. The smectic ordering potential is then deduced from the Boltzmann factor for the probability of finding a particle at position z . The diffusion coefficient in the smectic phase can then be calculated taking D0 as the diffusion coefficient in the nematic phase close to the N-Sm transition. 1987. Onsager.. A. 692. Mulder. The diffusion rate complies with the rate in the nematic phase. Therefore the diffusion in the smectic phase can be effectively considered as a one-dimensional diffusion of a Brownian particle in a periodic potential in the high friction limit. 99. as plotted in Fig. 627. we conclude that the smectic phase of semi-flexible hard rods consists of layers of glassy rods rather than fluid layers. t) can be directly obtained from the histogram of particle positions after a time t. Lett.68 to 0. 53 . which can be determined experimentally from the distribution of particle positions with respect to the middle of a layer parallel to the director. using this factor the MSD in the smectic phase is obtained from the MSD in the nematic phase. Phys. In conclusion.The use of such a potential is very common due to its simplicity [4]. Lett. The perpendicular motion is in all cases strongly subdiffusive: after the nematic-smectic (N-Sm) transition it reduces from 0. t) shows distinct peaks exactly at integer multiples of the particle length (and therefore of the layer thickness.44. This allowed us to give a full and coherent description of the diffusion process without any assumptions on the system. Helfrich. t). Perpendicular diffusion at high volume fractions is only possible through a subdiffusive reptationlike motion along the long axis to escape the local excluded volume and thus also hindered by the ordering potential. length. Rev. Lettinga and E. Acad. we have for the first time visualized the process of permeation in the smectic phase at the scale of single particles for a system of charged rods. 3(a)). 71. Thus the diffusion coefficient is predicted to decrease by a factor of 0. G(z. 197802. The anisotropy in the total diffusion.e. taking into account the ordering potential.. The effect of the ordering potential is also obvious with respect to the overall mean square displacement (MSD).Y. At high frequencies. video correlation spectroscopy. while the characteristic time for melting and forming is fast (of the order of a second) in the Df -state (where the subscripts “s” and “f” stand for “slow” and “fast”. On the basis of the different observed optical morphologies. the nematic phase of fd-virus suspensions is found-to be chiral-nematic (in the absence of an electric field). In the current study. a chiral nematic N * -phase is induced. the chiral texture disappears and the small N-domains melt and reform. Dhont IFF-7: Soft Condensed Matter We explore phase/state transitions in suspensions of charged fibrous viruses (fd) at low ionic strengths. There is a gradual transition to the ND * -phase at higher amplitudes at low frequencies. respectively).[1]). where the N-domains become smaller and disconnected and smaller sizes (see the third image in Fig. the equilibrium state is a nematic in coexistence with an isotropic phase.1 is a snap shot of a dynamic state.1. The phase/state transitions that are considered here are the result of interactions between external field-induced double layers.1 (see also Ref. Kang and Jan K. a much lower TRIS/HCL buffer concentration is used. 1: The electric phase/state diagram of fd-virus with a concentration of 2. where the relatively large Debye length screens the chiral nature of the core of fd-particles to an extent that renders the nematic phase non-chiral. as can be seen from the striped patterns in the second image in Fig. The fourth image in Fig. Birefringence measurement shown that in this H-phase the rods are “homeotropically” aligned [3]. phase/state diagrams are constructed in the applied field amplitude versus frequency plane.[2]).0 V/mm). E0 8 [V/mm] 6 4 2 0 0 10 [fd]=2. The phase/state diagram in the field-amplitude versus frequency plane is given in Fig. electric birefringence and small angle dynamic light scattering. At low applied field amplitudes (smaller than about 1.1). the depolarized image is uniform.0 mg/ml Df H Ds * ND N* N 10 1 10 2 10 ν [Hz] 3 FIG.0 mg/ml. coexistence between a non-chiral nematic and an isotropic state is (see the first depolarized microscopy image in Fig.. the dynamics of melting and forming is slow. larger than a few kHz. This phase is referred to as the N-phase. On increasing the field amplitude.1). Without an applied electric field.Electric Phase/ State Diagrams of Charged Fibrous Viruses (fd) K. the ionic strength is significantly affected by carbon dioxide that dissolves from the air (this is discussed in detail in Ref. 54 . This is the result of the chiral structure of the core of fd-virus particles. G. In existing literature. induced by external electric fields at low frequencies where double layers are polarized. The various phases/states and the characterization of transition lines have been investigated by pitch measurements. Several phases/states are induced at frequencies below a few kHz. while a uniform aligned phase is observed at higher frequencies. In the dynamical Ds -state. At these low buffer concentrations. On further increasing the field amplitude. CV ( t ) = CV for four field amplitude at a fixed low frequency (the numbers are in V/mm). The optical-pitch variation is measured from optical images up to the N * .1 is not corrected for electrode polarization. 2008 [2] K. A. Interestingly. with a degree of orientational order that is essentially independent on the field amplitude and frequency.G. but also the domain size [1] K. Phys. which decreases with increasing amplitude to a saturation value of about 12 μm .5 3. The corrected phase/state diagrams can be obtained from this electrodepolarization theory [3].ND * -transition line.09 5. especially in the H-phase.K. E. Phys. diverges. J. J.2 shows a few correlation functions together with fits to a singlestretched exponential.and Df -states is characterized by means of video correlation functions CV . Dhont.0 CV τ[s] 15 Df ND * 0. For low amplitudes. 214501. The pitch is observed to diverge at the N * . the largest measured optical π -pitch is the true pitch.78 3. I is the transmitted intensity at a given pixel and the brackets <…> denote averaging over all pixels. defined from the transmitted light intensity time traces as recorded by a CCD camera (a snap shot is given in the fourth image in Fig.G. Wilk. It is even an open question whether polarization of condensed ions may play a role (about 85 % of the bare charge on fd-virus is screened by condensed ions). Dhont.Ds transition line This is probably due to the fundamentally different microstructural ordering in the ND * -phase and the Ds -state. Dhont. On further increasing a field amplitude at low frequency. Patkowski. the microscopic dynamics as probed with small angle dynamic light scattering is discontinuous at the ND * -to. In Ref.1 where several transition lines meet. A.Ds transition line. Electrode polarization arises from the formation of double layers at the electrodes that partly screen the applied electric field.K.1). dynamical states appear: The dynamics of melting and forming of N-domains in the Ds .[3] we derived an expression for the attenuation factor (the ratio of the bulk-value of the electric field and the applied field amplitude). 126. Kang.77 0 0. 84. resulting in different mobilities of the fd-viruses At the point in the phase-state diagram in Fig. 2008 55 . Lett. These measurements reveal that the rods are aligned along the electric field. Since the 2D projection of the optical pitch is measured. while at higher field amplitudes the director tends to be perpendicular to the external field. thick electric double-layers are probably responsible for the observed phases and states. Inter-colloidal interactions of fd-virus particles through polarized. As can be seen. The plot on the right in Fig. The left plot in Fig.26 10 5 Ds 4. J. 14005.An electric cell is used to characterize several phases and states by various optical techniques: Electric birefringence is used to detect alignment and the degree of orientational order. There thus seems to be an analogous “critical behaviour” as in equilibrium systems.G. Rev. [3]. the time constant diverges on approach of the ND * -to.ND * -transition line on variation of the field amplitude. Kang. The solid lines are fits to a single stretched exponent.0 3 4 5 0 10 20 30 tim e [s] 6 7 E0 [ v/mm ] FIG.2 shows the time constant for melting and forming as a function of the field amplitude. not only the time constant for melting and forming of N domains diverges. Eur. 2007 [3] K. J. This expression shows that electrode polarization can be neglected for our electrical cell for frequencies above 100 Hz. Right: The characteristic time constant of melting and forming of small N-domains as a function of the applied field amplitude. The large spread near the N * - 1. the measured chiral π -pitch shows a quite large spread. [ I ( t ) − < I ( t ) > ][ I (0) − < I (0) > ] [ I (0) − < I (0) > ]2 Here. 2: Left: Video correlation functions ND * -transition line indicates that the director attains arbitrary values. Within the Df state the time constant is essentially independent of the field amplitude. Kang. Chem.K. Submitted to Phys. So far there is no theory that could explain the observed phase/state behaviour. The phase/state diagram in Fig. Wiegand IFF-7: Soft Condensed Matter The molecular origin of the thermal diffusion process or Ludwig-Soret effect is one of the unsolved problems. colloidal particles show many physical phenomena that are also found in ordinary molecular systems. in some cases. colloids have been used frequently to study fundamental questions in physics. The temperature dependence of the Soret coefficient is mainly determined by single particle contributions and agrees to some extent with the temperature dependence of the surface coating material. Recent advancements open up alluring perspectives to exploit thermophoresis as a novel tool in microfluidic manipulation. in toluene. and selective tuning of colloidal structures. for several aqueous mixtures with and without solutes such as polymers and colloids. even qualitative predictions. modern optical techniques have been developed which allow the investigation of complex fluids with slow dynamics such as polymer solutions and blends. Schematic illustration of the thermal diusion process in a binary mixture in a temperature gradient. octadecane. A pronounced concentration dependence of the Soret coefficient has been found in experiments [4. respectively. Being just very large molecules in a solvent. Usually. Therefore. The scenario for a binary mixture of particles is sketched in figure 1. the mechanism leading to a sign change is system dependent. We studied a colloidal dispersion in the intermediate concentration range (volume fraction φ < 10 %) and found that the interactive part of the Soret coefficient agrees with an analytical theory.Thermal diffusion behavior of hard sphere suspensions H. where long ranged repulsion between charged micelles and colloids has been considered [1]. Although. S. FIG. Also the temperature dependence of ST for a large class of macromolecules and colloids in water shows a distinctive universal characteristic [1]. isotope separation). describes the diffusive mass transport induced by a temperature gradient in a multicomponent system. In dilute solutions. A number of recent studies show that interactions play an important role for the thermal diffusion behavior. which was discovered already 150 years ago. where colloid-colloid interactions can be neglected. 1: In recent years. One of our strategies to tackle this problem is the investigation of a spherical colloidal model system with a short range repulsive interaction potential. Buitenhuis. we found the sign change concentration is almost system independent and strongly correlated with the breakdown of the hydrogen-bond network [3]. This effect. colloidal disper- 56 . characterization of geochemical processes (Salton Sea geotherm. it is expected that they are also a suitable model system to illuminate the microscopic mechanism underlying the Ludwig-Soret effect. also known as thermal diffusion. thermal diffusive behavior of highly diluted and concentrated solutions can be differentiated. The small and big particles accumulate at the hot and cold side. oil reservoir composition) and combustion. Ning. 1] and is predicted by theory [5]. which are of practical importance in separation processes (Thermal field flow fractionation of polymers and colloids. the thermal diffusion coefficient of the colloids is determined by the nature of the interactions between single colloidal particles and solvent molecules (and possibly other solutes like ions that form a double layer around the colloids) [2]. Conceptually. At higher concentration the Soret coefficient follows a power law. Structural changes of the surrounding solvation layer due to temperature changes and/or changes of the solvent composition may induce a sign change of the thermal diffusive behavior of single colloidal particles. Consequently. It relates to our poor understanding of non-equilibrium statistical mechanics pointing out our incapability of obtaining. J. micellar solutions. Colloidal particles are small enough to exhibit thermal motion commonly referred to as Brownion motion. [7] J. with a radius of a = 27 nm dispersed in toluene. Kita. while under good solvent conditions the particles move to the cold side. H. sterically stabilized by octadecane. 57 . that if the particle coating changes from " organophilic" to "organophobic" a sign change could be expected. The main issues of interest were the derivation of scaling laws and to understand the sign change of the Soret coefficient for macromolecular and colloidal systems on the basis of existing theories for molecular fluids. Ning. In the past few years several theoretical concepts have been proposed to understand single particle and colloid-colloid interaction contributions to the thermophoretic motion of colloidal particles [1]. In general one can expect. Typically. but under which conditions this is the case needs to be clarified in further investigations. Wiegand. J. a sign change of the Soret coefficient as a function of temperature and concentration is possible for appropriate interaction parameters. [5] J. while the single part shows a strong temperature dependence. Also the fact that octadecane is bound to a surface might influence the thermal diffusion behavior. 2 shows the Soret coefficient as function of the volume fraction φ for different temperatures. Phys.K. Buitenhuis. J. But for the low molecular weight system no sign change occurs in the investigated temperature range. the movement of the solute particles to the warm side is an indication for poor solvent conditions. 32. Chem. FIG. Wiegand. Luettmer-Strathmann. and R. Dhont. 61 (2008). Eur. J. According to the theoretical approach by Dhont the single ( ) and the collective part ( ) of ST can be separated. J. the work by Dhont gives explicit expressions for the contribution of colloid-colloid interactions to the thermal diffusion coefficient DT . R. Phys-Cond. At high concentrations ST follows a power law as it also has been found for polymers approaching the glass-transition [7]. This might be an indication that also the silica core of the particles influences the thermal diffusion behavior. Phys. Ning. Ning. At low temperature the colloids move to the warm side. The solid line represents a t of the data according to the theory by Dhont [5]. 153102 (2008). These interaction contributions lead to a concentration dependence of the thermal diffusion coefficient. J. [3] S. Non-Equilib. [6] H. 10746 (2006). Piazza. In the intermediate concentration range the concentration dependence of the Soret coefficient can be described by the theory for hard spheres [5]. For the investigated system the vicinty of a gel line at low temperatures is probably responsible for movement of the colloids to the warm side. The inset shows ST of octadecane in toluene versus temperature for a octadecane concentration of w=5 wt%. Kriegs. Although the exponent is two orders of magnitude smaller. Kita. 1632 (2004) and 1642 (2004). [4] H. while at high temperatures the colloids move to the cold side. 3). FIG. 110. 120. and S. The thermal diffusion behavior was studied in a concentration range between 1% and 30% by volume fraction and in a temperature range from 15-50 ◦ C [6]. We studied spherical silica particles. Parola. E 25. Dhont. Fig. Wiegand. 3571 (2005). shows. Phys. Phys. Macromolecules 38. 204911 (2006). For intermediate temperatures a sign change of the Soret coefficient occurs for higher concentration. and S. While the majority of the theoretical approaches give expressions for the single particle contribution. G. Köhler. 125. Dhont. According to this theory. B. K. Chem. Rauch and W.G. J..sions and bio-molecules. J. 193 (2007). J. The dashed lines are guides to the eye. J. [1] R. H. Matt 20. Chem. A. Thermodyn. 3: Concentration dependence of the Soret coecient at dierent temperatures. K. which has the same tendency as the Soret coefficient of octadecane in toluene (cf. Fig. that the interactive part is almost temperature independent. [2] J. G. 2: A separation of the Soret coefficient into a single particle contribution and an interactive part. The measured Soret coecient (•) versus temperature for a colloidal suspension with a volume fraction of φ=10%. 58 . Our technical repertoire reaches from the analysis of proteins using molecular biological and biochemical methods to physiological studies in vitro and in vivo in normal and in transgenic animals. single molecule fluorescence spectroscopy. Signal transduction usually begins with receptor proteins that register a stimulus and start a cascade of biochemical reactions which often ends with the opening or closure of ion channels that changes the electrical properties of the cell membrane. Frank Müller The Institute of Structural Biology and Biophysics. 59 . Moreover. G-Protein-mediated signalling as well as mechanisms of excitation and adaptation of sensory cells and neurons are investigated. chemical and physical stimuli evoke characteristic physiological responses. signal transduction and information processing are studied at different levels of complexity: on the molecular level we study the properties of receptor proteins and ion channels. we study information processing in small well-characterized neuronal circuits. and camera-based ratiometric imaging setups. and physicists. A strength of the ISB-1 has always been the tight co-operation of biologists. In every cell. information processing. imaging techniques have been estab-lished. including twophoton fluorescence-lifetime imaging microscopy. in order to adapt to changes in the environment each cell must constantly modulate these signalling processes. The cell is the smallest living unit. Both synthetic fluorescent dyes as well as genetically encoded sensors based on fluorescent proteins are employed in the institute. The analysis of cellular processes with high spatial and temporal resolution provides the basis for an in-depth understanding of the “physics of the cell”. On the cellular level. signal transduction. In ISB-1. On the network level. and cell bio-physics. chemists. Fluorescence-based optical methods and imaging play an important role in the research of ISB-1. Cellular Biophysics ISB-1 (formerly INB-1 or IBI-1) is dedicated to research in molecular and cellular neurobiology. To monitor the concentration of intracellular messengers like calcium or cAMP. such as the retina or the olfactory bulb.ISB-1: Cellular Biophysics Acting Director: Prof. 7 (n = 8) and 55. Klin. Kirchhof2. We generated knock-in mice that harbour a single amino-acid exchange (R669Q) in the cyclic nucleotide-binding domain of the HCN4 channel that abolishes cAMP binding to the protein. Immunologie. E. In adult mice. HCN4 channels take no longer part in heart rate regulation. and 60 . B.Univ.1 mV (n = 5) for HCN4 . 2Med. The If current.H. HCN4 is the principal cardiac pacemaker and persistent elevation of the heart rate by cAMP is essential for viability.7 mV (n = 3) for HCN4. Voltage-dependent activation of R669Q heterologously expressed HCN4 (circles) and HCN4 channels (squares). Med. Prior to E12. Upon activation of adrenergic receptors. 1: Electrophysiological characterization of HCN4 and R669Q HCN4 channels. und Poliklinik. A. Kaupp1 1 3 ISB-1: Cellular Biophysics.3 ± 3. Münster und IZKF Münster. Mainz. P. K. HCN channels are unique in that they are activated upon hyperpolarization rather than by depolarization of the membrane potential. Voltages of half-maximal activation (± SD) in the absence (open symbols) and presence (filled symbols) of cAMP (100 µM) were -79. Harzheim1. The depolarizing current flowing through HCN channels. Within the superfamily of voltage-gated channels. plays an important role in controlling cardiac rhythmicity. homozygous and heterozygous embryos display reduced heart rates and show no or attenuated responses to catecholaminergic stimulation.4 (n = 7) R669Q and -81. The arginine residue is crucial for the binding of cAMP. whereas no shift was observed for the HCN4R669Q mutant (Fig. To ascertain that the mutation abolishes regulation by cAMP. Zürich. Kremmer3. Spontaneous activity of the mammalian heart is generated in the sino-atrial node (SAN). The question how pacemaker activity is generated in SAN cells and how pacemaking is regulated by the autonomouos nervous system is still a matter of debate. Fabritz2. Native HCN channels consist of four subunits. because it interacts with the negatively charged phosphate group. Fig. hearts exhibit pauses and sino-atrial node block. Univ. We analyzed the spontaneous beat frequency of +/+ +/R669Q hearts isolated from HCN4 . the mechanism of pacemaking may switch during development and HCN4 may serve two different functions that critically rely on the presence of cAMP. These results suggest that in the embryo. D-48129 Münster Helmholtz Zentrum München. respectively. Cyclic AMP shifted the activation curve of the wild-type HCN4 channel by +25 mV. In mammals four genes (HCN 1 – 4) have been identified to encode individual subunits.5 ± 3. Waisman5. HCN4 . Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are thought to play a major role in cardiac pacemaking. voltagedependent opening of these channels is regulated by the direct binding of cAMP to the polypeptide.The Role of HCN4 channels in Cardiac Pacemaking D. R. Buch4. Homozygous R669Q/R669Q mice die during embryonic HCN4 development. In this study. Pfeiffer1. & Poliklinik.6 ± 2. 1). and -84. Adult heterozygous mice display normal heart rates at rest and during exercise. We studied the role of If in mice. D-55131 Mainz Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated ion channels. therefore. Univ. L.3 ± 7. we decided to investigate the precise role of HCN4 in cardiac pacemaking of mice. In addition. Seifert1. D-81377 München. T. The voltagedependent activation of HCN4R669Q mutant and wild-type channels is similar. we analyzed the wild-type and the HCN4R669Q mutant after heterologous expression in a cell line. mediated by HCN channels. leading to a higher open probability of HCN4 channels. cAMP levels increase. designated as Ih or If. HCN channels are important targets for adrenergic stimulation. but prevent sinus pauses during and after stress. U. in which binding of cAMP to HCN4 channels was abolished by a single amino-acid exchange (R669Q) [1]. is activated at negative membrane potentials. However. following βadrenergic stimulation. If leads to excitation of the cell and spontaneous firing of action potentials and is. Moreover. 4Exp. Thus. CH-8057 Zürich 5 I. thought to be one of the primary ionic mechanisms generating cardiac spontaneous rhythmic activity. Basal heart rate during embryonic development +/+ +/R669Q (E9. 3: HCN mice display a sino-atrial block. 0/9 +/+ HCN4 hearts). in wild-type embryos. 15235 (2003) 61 . Herrmann. HCN4 : red.8 %. Waisman. 3. consistent with sinoatrial block. perfused hearts.5 was identical. 2). however.5 ± 5 pauses per 55 minutes). 2).H. Pfeiffer. +/R669Q Our results show that only in embryonic mice. Sino-atrial block and sinus pauses were also found in spontaneously beating. Under basal conditions. Notably. Stieber. Furthermore. Natl. Harzheim. In adult mice. the heart rate whereas in HCN4 is significantly reduced (Fig. during orciprenaline infusion: pauses in 5/10 HCN4+/R669Q vs. whereas no increase was observed in hearts from HCN4R669Q/R669Q embryos (2. HCN4 channels are probably no longer involved in sympathetic stimulation of the heart rate. a. The WT heart shows a +/R669Q constant sinus rhythm. b. Seifert. This increase was no longer present in HCN4+/R669Q embryos R669Q/R669Q embryos. Kaupp and R. Fabritz.Kirchhof. After +/R669Q mice developed pauses and exercise. Biel. Langendorff- Fig. 3.8 %). M. HCN4 channels serve as pacemaker controlling the heart beating frequency. nor was the electrophysiology of the atrio-ventricular node and the ventricle. demonstrating that the point mutation in the HCN4 channel has the same effect on the basal heart rate as the complete loss of the channel [2]. These but not from HCN4 results demonstrate that HCN4 is the principal target for cAMP during the embryonic stages of mouse development. a right atrial electrogram (RA). 27. K. mostly during washout of the βadrenoceptor agonist orciprenaline (Fig. HCN4 sino-atrial block more often than their wild-type littermates (Fig. Acad. Kremmer. Hofmann and A.HCN4R669Q/R669Q embryos prior to E11. EMBO J. [2] J. R669Q Isoproterenol superfusion increased the rate of isolated hearts from HCN4+/+ and HCN4+/R669Q embryos (~ 37. channel alters the embryonic heart Fig. the heart rate was significantly slower compared to hearts from wildtype embryos (Fig. The number of embryos analyzed HCN4 is indicated.B. 2: The HCN4 beat. HCN4 : green. Representative recording of spontaneous rhythm in isolated. A. Buch.5. Representative examples of telemetric ECG recordings in freely moving mice carrying an implanted ECG transmitter.5-11. S. The heart rate of sedated animals and the intrinsic rate of the isolated heart was also not different between genotypes. the beat frequency of hearts from HCN4R669Q/R669Q and HCN4-/. L. Shown is a ventricular monophasic action potential (MAP). however. increased the heart rate from HCN4+/+ and HCN4+/R669Q embryos.4 % and 19. the HCN4 heart shows three pauses with doubling of cycle length. Sci. 1/9 HCN4+/+ hearts. this activity is severely affected when cAMP modulation of the channel is impaired. the channels may serve a back-up mechanism that maintains a stable heart beat in situations during and after stress and. Here. the contribution of HCN4 channels is less pronounced. Löster. hearts from heterozygous and homozygous embryos beat regularly without obvious arrhythmias. S. P. an activator of membrane-bound adenylyl cyclases. Animal numbers are indicated. R. All recordings were obtained during the 55 minute recovery period after stress tests (air jets or swimming). Proc. J. The differences in basal heart rate and in the activation of If between wild-type and heterozygous embryos prompted us to study the heart rate in freely moving adult HCN4+/+ and HCN4+/R669Q mice by telemetric recording of ECG. the heart rate increased during E9 to E11 of embryonic development (Fig. Notably. during washout of orciprenaline: pauses in 8/10 HCN4+/R669Q vs. superfusion with NKH477. and lead II of the tissue bath ECG (ECG). respectively). F. Neither the heart rate at rest nor during exercise or mental stress was different between genotypes. Feil. E. HCN4 : 6. Langendorffperfused hearts. T. 692 (2008). HCN4+/R669Q: 9 ± 7 pauses per +/+ 55 minutes. Similarly. 1/9 HCN4+/+ hearts. Feil. 2). pauses in 4/10 HCN4+/R669Q hearts vs. R669Q/R669Q embryos. USA 100. [1] D.embryos at E9. U. Ludwig. R669Q/R669Q : blue). (A) HCN1-specific antibody labels photoreceptor inner segments.B. inner plexiform layer. thereby. Univ. 3]. D-35390 Giessen In the mammalian retina. U. M. play an important role in the integrative behaviour of neurons and the sensitivity to synaptic input. Ophthalmology. Giessen and Marburg. HCN channels become activated during hyperpolarization in bright light and depolarize the cell toward the dark membrane potential. Friedburg3. In the outer retina. (D) Double staining against CabP5 (left) and HCN1 (right) shows that HCN1 is present in the plasma membrane of cones (arrow). Shaping of voltage responses by the kinetic properties of the ion channels involved in the generation and propagation of electrical signals. Some ganglion cell somata are also HCN1-positive in the GCL. Fig. D-72076 Tübingen 3 Dept. Lateral inhibition within the neuronal network involving feedforward and feedback mechanisms and 2. outer plexiform layer. inner nuclear layer. HCN channels were suggested to shape the light response. G. Tanimoto2 1 2 ISB-1: Cellular Biophysics Inst. Univ. Representative records of a wild-type mouse are 62 . Kaupp1. IPL. are depolarized in the dark. Light responses are prolonged in HCN1 knock-out mice The murine Ganzfeld ERG is a measure of the overall retinal function. we show that HCN1 is particularly strong expressed in both rod and cone photoreceptors. which are activated by hyperpolarization and gated by cyclic nucleotides. IS. 1: Immunohistochemical localization of HCN1 in the mouse retina. outer nuclear layer. rod photoreceptors are strongly HCN1-immunoreactive. GCL. the light response of photoreceptors is processed by an elaborate neuronal network. we show that HCN1 channels shorten retinal light responses in both rod and cone pathways [1]. Ophthalmic Res. OPL. cellular signals are shaped by two principal mechanisms: 1. N. rods and cones. HCN1 is strongly expressed in the mouse retina HCN1 is expressed throughout the retina. Wild-type and HCN1 knock-out animals were littermates (six weeks of age). A. Somata of certain bipolar cells (long arrow) and amacrine cells (short arrow) are labelled in the INL. the output neurons of the retina. outer segments. F. Mataruga1. Along the way from photoreceptors to ganglion cells. somata. In mammals. (B) No HCN1 immunoreactivity is observed in retinal sections obtained from HCN1 knock-out animals. At least ten types of cone bipolar cells and one type of rod bipolar cell provide the pathways for the signal flow from photoreceptors to ganglion cells. OS. Tübingen. By recording scotopic and photopic light responses in normal mice and in HCN1 knock-out mice. making the light response transient. INL. The photoreceptors. Thiel1. One family of voltage-gated channels present in the retina are the HCN channels.Knop1.W. (C) CabP5-immunoreactivity is detected in a subset of cone photoreceptors. C. ONL.Light Responses in the Mouse Retina are Prolonged Upon Targeted Deletion of the HCN1 Channel Gene F. Seeliger2. Cone photoreceptors that can be labelled with antibodies against the calcium-binding protein CabP5 are also positive for HCN1. corresponding bands of bipolar cell axon terminals (long horizontal arrow) and amacrine cell dendrites (arrowhead) are visible in the IPL. HCN channels affect the cable properties of dendrites and shape the time course and propagation of excitatory and inhibitory postsynaptic potentials. Here. ganglion cell layer. We performed single-flash ERG recordings under scotopic and photopic adaptation conditions and with different stimulus intensities. inner segments. four channel genes (HCN1-4) have been identified. axons and axon terminals in the outer retina. but hyperpolarize upon illumination. HCN channels co-determine the resting potential and membrane conductance and. Müller1. In rod and cone photoreceptors. We have previously shown that HCN channel isoforms are differentially expressed in the retina [2. Mataruga. at higher flash intensities like 300 mcd*s/m² that caused a manifest prolongation of the responses. Müller. i.B. Müller. presumably because the hyperpolarization of the photoreceptors was too small to activate HCN1 channels. J.B. 17. there were no substantial differences between wt and knock-out mice. (B) At higher flash intensities like 0.shown in the left columns of Fig. Eur. N. followed by a large positive-going component. Direct comparison of flash ERG recordings in dark-adapted (A) and light-adapted (B) wild-type (Wt) and knock-out mice -/(HCN1 ).C). 2221 (2008) [2] E. A. In the statistical analysis (Fig. the b-wave is dominated by the activity of ON-bipolar cells. (A) At low flash intensities like 0.e. the photopic (light-adapted) responses are reduced and their oscillatory components also diminished (Fig. Scholten. ERG waveforms consist of an early negative-going component best visible at high light intensities. the b-wave. Under both scotopic and photopic conditions.and b-waves (Fig. Kaupp. it is tempting to speculate that already at the level of the retina the flicker fusion frequency. Haverkamp. Vis. Fig. Eur. Thiel. Knop. light responses are significantly modulated. 3: Effect of HCN1-deficiency on scotopic ERG flicker fusion frequency. i. Indeed. the knock-out of HCN1 channels had no substantial effect on the onset of the ERG response or the amplitudes of the a. Typically. the flicker data were rather similar. the photoreceptor. J. Kremmer and U. n= 4 for both wild-type and HCN1 knock-out animals).01 cd*s/m². Friedburg. Müller. small wavelets that probably involve inner retinal circuitry. Our results show that already in the first cell of our visual system. (D) Statistical evaluation of the corresponding ERG b-wave data. Neurosci.3 cd*s/m². should be reduced. The normal range is delimited by black lines indicating the 5 % and 95 % quantile of the wild-type data. whiskers the 5 % and 95 % quantiles and the asteriks the median -/of the HCN1 data.C. Flicker detection is compromized in HCN1 knockout mice As single flash responses are prolonged.W. light responses in wildtype and knock-out animals were similar. In contrast. Seeliger. Boxes indicate 25 % and 75 % quantile range. 2A (scotopic) and B (photopic). [3] F.e. Whereas at least the initial portion of the a-wave reflects the primary light response in photoreceptors. 2A. 63 . Ivanova. A. M. C. E. the b-wave was considerably prolonged in HCN1 knock-out animals at higher light intensities. 2: Effect of HCN1-deficiency on retinal function under scotopic and photopic conditions. U. Neurosci. Neurosci. Riding on top of the bwave are the oscillatory potentials. 2084 (2003). the apparent prolongation of the waveform led to a marked reduction in the ability to follow high frequency stimulation. 143 (2006). F. At low flash intensities. 23. S. the a-wave. The following figure compares ERG recordings in wildtype and HCN1 knock-out animals to repetitive light stimuli at different frequencies and different light intensities. (C) Superposition of recordings from wild-type and knock-out animals reveals that light responses are considerably prolonged in knock-out mice. the ability to respond to repetitive stimulation. The most prominent effect of HCN1 knock-out concerned the duration of light responses. C). Tanimoto and F. Ivanova and F. [1] G. 28. In contrast. Kaupp. 2D. the scotopic b-wave amplitude data recorded from HCN1 knock-out animals (box plots) fall well within the black lines that limit the 5% to 95% normal range of the wild-type data. Fig. 2B. E. at 10 mcd*s/m². there was a remarkable reduction of the ability to follow higher frequency flicker in the knock-out animals. In the scotopic regime. The amino-acid sequences were obtained from NCBI databases RnAC1 . 64 . Mujagic3. S. 1) that showed the closest relationship between AmAC3 and AC sequences from rat olfactory neurons (RnAC3. [3]). S. the concentration of the intracellular messenger cAMP increases. and C. As a result of AC-activity. TU Berlin. A dendrogram was constructed (Fig. The scale bar allows the conversion of branch lengths in the dendrogram to genetic distance between clades (0.g. AC1 from Rattus norvegicus (Rn). MmAC8. The enzymatic properties of AmAC3 were determined after heterologous expression of the gene in a cell line. CeACY2. 1: Phylogenetic comparison of AmAC3 with mammalian. this gene is orthologous to the mammalian olfactory AC type 3. CeACY3. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. The same rank order of inhibitory potency was shared between AmAC3 and ACs in the antennal lobes of honeybee brain. AC2 from Mus musculus (Mm). RnAC5. motor and higher-order information processing in the honeybee brain. The catalytic domains of the enzyme reside in the cytosolic loop connecting the M1 and M2 domains and in the cytoplasmic C-terminus. RnAC6. DmAC10B. D-14476 Golm 3 Institute of Ecology. AC1 from Caenorhabditis elegans (Ce). CeACY1. Schlenstedt2. Gauss1. The sequence of the soluble MmAC10 was used as out-group. A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. AC1 from Drosophila melanogaster (Dm). This messenger regulates and modulates the activity of protein kinases. MmAC2. Fig. Membrane-bound ACs (tmACs) share a common and characteristic topology with two large hydrophobic membrane domains (M1 and M2) within which the amino-acid chain spans the plasma membrane six times. MmAC7. RnAC3. All tmACs are activated by α subunits of stimulatory G-proteins (Gsα). R. cloned and native ACs. J. Wachten1. MmAC9. Recently. DmAC39E). The numbers at the nodes represent the per cent bootstrap support for each branching. [5]). Therefore. were in the low micromolar range. in sensory signal transduction. Values for half-maximal activation with a water-soluble analogue of forskolin (NKH477) of both. Production of cAMP is based on the activity of adenylyl cyclases (ACs). DmAC35C. Potsdam. RnAC4. Baumann1. We have now compared the biochemical and pharmacological properties of heterologously expressed AmAC3 with native ACactivity in identified subregions of the honeybee brain [2]. J. Molecularly. cardiac myocyte regulation. we performed a phylogenetic analysis of the derived amino acid sequence to a variety of vertebrate and invertebrate AC sequences. Our results suggest a role for AmAC3 in sensory. Fuss1. N. DmAC76E. [4]) and the Drosophila protein (DmAC39E.Molecular and Functional Properties of Native and Heterologously Expressed Adenylyl Cyclases A. DmACrutabaga. we cloned the first gene from the honeybee (Apis mellifera) encoding a membrane-bound AC (Amac3) [1]. DmAC78C. Erber3 1 2 ISB-1: Cellular Biophysics Institute of Biochemistry and Biology. changes of the cAMP concentration can have a significant impact on cellular signaling behavior. Drosophila.a. After cloning the Amac3 gene from honeybee brain. Most tmACs are also activated by the diterpene forskolin or NKH477 (s. Univ. ion channels as well as transcription factors. elegans ACs. CeACY4. This small organic compound participates e. as well as in learning and memory.1 = 10% genetic distance). following the M2 domain. D-10587 Berlin Cyclic AMP serves as an important intracellular messenger in virtually all organisms. Signal. motor and even higher brain functions. 6x106 4x106 2x106 0. 12. Science. a cell line was generated (Fig. This CNG channel is permeable to Ca2+ ions. 2+ 15 µm The distribution of Amac3 transcripts and protein in honeybee brain was examined by in situ hybridization and immunological staining [1. if not identical properties. Therefore. 125 (2000). J. 1495. Biochim. 2]. These results have been submitted for publication recently [2]. Fig. Erber. Reed.2 µM and 3.R. Baumann. Fluorescence [counts / mg total protein] 8x106 flpTM flpAC3 6x106 4x106 2x106 0. HA-tagged AmAc3 was visualized using a rat anti-HA-antibody and a fluorescently labeled secondary antibody (goat-anti-rat ALEXA 568). Neurochem.and flpAC3 cells evoked by AC stimulation with increasing forskolin concentrations (0. a specific agonist of tm-ACs (Fig.A. Acta. Biophys. Data are mean values of two independent experiments performed in duplicate. Bakalyar and R. S. Wachten and A. the cell lines were used to monitor the G-protein coupled receptor mediated activation of AmAC3. 2) that constitutively expresses AmAC3 and a cAMPgated ion channel (CNG). AmAC3 may serve in vivo to translate GPCR. 250. Above: Fluo-4 signals of flpTM. 3).1 1 Norepinephrine [µM] 10 100 65 . Therefore. 1403 (1990).In order to determine the enzymatic properties of AmAC3. 2: Immunocytochemical detection of heterologous expressed AmAC3. R. Chern. S. Fuss. Cell. [2] N. an increase in 2+ AmAC3 activity can be monitored by Ca -imaging due to the subsequent activation of the CNG channel. Relative fluorescence intensities are given in counts / mg total protein. [3] Y. J. 195 (2000). We used this cell line as well as a cell line that only expresses the CNG channel to determine the dose-response curve of AmAC3activation by forskolin.01-30 µM). Levin. Error bars indicate the standard deviation. (Left) Light-microscopic photograph of a cell.and flpAC3 cells evoked by AC stimulation with increasing norepinephrine concentrations (0. [5] V. Baumann. The AmAC3 protein is located in the plasma membrane (arrows). 3). Gauss and A. Fluorescence [counts / mg total protein] 8x106 flpTM flpAC3 [4] H. In both cell lines ßadrenergic receptors are expressed endogenously. Wachten. 96.01 0.1 1 10 Forskolin [µM] 100 rel. 3: Effects of AC activation on Ca -dependent Fluo-4 signals in AmAC3-expressing (flpAC3) and parental (flpTM) cells. Fig. Mujagic. (Right) The same cell viewed under fluorescence-microscopic conditions.R. Iourgenko and L. Schlenstedt. The EC50 values for activation of AmAC3 by forskolin and norepinephrine were 15. we analysed the biochemical and pharmacological properties of native and cloned enzymes. Below: Fluo-4 signals in flpTM. J.and/or Ca2+-mediated signals into changes of the intracellular cAMP concentration and thus to participate in sensory. Using protein preparations of subregions of the honeybee brain and from the AmAC3-expressing cell line. [1] S.1 µM (Fig.1150 µM). The prominent expression of AmAC3 in the antennal lobes of honeybee brain suggests AmAC3 to participate in the processing of olfactory signals. Adenylyl cyclases expressed in the antennal lobes of honeybees and AmAC3 share very similar. 1580 (2006). rel. In addition. submitted. The NBD-group is a polarity sensitive molecule.5 µM) and cAMP (closed circles) or cGMP (open circles) for binding to the mlCNG protein (0.7 nM for cAMP and 300. In contrast. R.e. CNG channels open and thereby cause changes in membrane potential. Previously. Cukkemane. 8-NBD-cAMP fluorescence in the absence of the mlCNG protein was subtracted. The solid lines represent non-linear least-squares fit. Gensch. Seifert. the binding of 8-NBD-cAMP to the mlCNG protein was competed with non-labelled cyclic nucleotides (Fig. The presence of a thrombin cleavage site allowed us. 2). (B) Competition between 8-NBD-cAMP (0. A fluorescent cAMP-analogue.7 µM for cGMP. the molecular events that relay ligand binding to channel activation are not well understood.Functional Studies of a Prokaryotic Cyclic Nucleotide-Gated Channel A. The solid line represents non-linear leastsquares fit. Upon binding of cyclic nucleotides. Here.6 ± 17. i.5 nM for cAMP and 320. The KD value was 81. Next we determined whether the binding properties of the tetrameric channel differed from the isolated CNBD. Both.9 ± 2.3 nM (cAMP) and 363. K. 1A).4 nM for cGMP for the wildtype CNBD. As binding and gating are intimately coupled.3 µM for cAMP. This allowed determining quantitatively the binding affinity of the channel for this ligand (Fig. The molecular interactions that tune affinity and selectivity of cyclic nucleotide binding are not fully understood.7 ± 25. we examined the ligand-binding properties of a K+-selective CNG channel that was cloned from the bacterium Mesorhizobium loti (mlCNG) [1].5 µM).3 ± 5.3 nM. The KD values obtained for the R348A mutant were 18. Ligand binding studies were performed on heterologously expressed and purified tetrameric mlCNG protein as well as on its isolated cyclic nucleotide-binding domain (CNBD).4 ± 15. Bönigk.3 µM). like epithelial exchange factor (EPAC).9 nM (cGMP). Novak. The KD values were 67. The data were analyzed assuming a simple binding model.5 nM for cGMP. Kaupp ISB-1: Cellular Biophysics Cyclic nucleotide-gated (CNG) channels play a fundamental role in signal transduction of sensory neurons. it is difficult to separate one from the other. B. cAMP and cGMP. We observed that cAMP co-purifies with wildtype CNBD. 8-NBD-cAMP. To determine the KD values of physiological cyclic nucleotides. We expressed both variants of the CNBD and purified the proteins on a glutathione affinity column. because ligand affinity has not been measured directly but rather has been inferred from electrophysiological studies. to purify the CNBD without its GST fusion partner. This property of the NBD group has been widely utilized in biophysical studies on lipids as well as on proteins. T. In order to perform binding studies on purified protein. Grüter. Ligand binding to purified CNBDs was studied using the same assay as for the intact channel (Fig. 66 .5 ± 4. (A) Normalized increase of fluorescence of 8-NBD-cAMP on binding to mlCNG protein (0. Although the physiological role of CNG channels has been extensively studied.e. was used to study ligand binding to mlCNG. 1: Ligand binding of the full-length mlCNG protein. Fig. To perform ligand binding experiments with the wildtype CNBD. i. This allowed purifying large amounts of the 2+ protein using a Co affinity column. U. it was necessary to unfold and refold the CNBD to obtain cAMP-free protein. The mean KD value for 8-NBD-cAMP was 15. It is largely non-fluorescent in aqueous solution and becomes fluorescent in a hydrophobic environment. B. R348A has no cAMP bound. We observed a pronounced fluorescence signal upon binding of 8-NBD-cAMP to purified mlCNG.8 ± 8. and 22. the mlCNG was expressed as a fusion protein with a hexa-histidine tag at its C-terminal end. the wildtype CNBD and a mutated version (R348A). These results indicate that the binding and gating properties of the bacterial channel are distinctively different from those of mammalian CNG channels. have been expressed and purified as GST-fusion proteins to determine their crystal structures either in the ligand-bound or ligand-free (R348A) state [2]. that has a much lower affinity. The KD values were 71. the mlCNG protein and the CNBD bind cAMP in a non-cooperative manner with similar affinity. W. 1B).7 nM. The KD value was 17. (D) Competition between cAMP and cGMP with 8-NBD-cAMP for binding to mutant CNBD (R348A) (3 µM). Although we could determine the KD values for cAMP and cGMP binding to the mlCNG channel in quantitative terms. ligand selectivity and sensitivity are affected by this single amino acid exchange and thereby foster the molecular understanding of the CNG channel activation process. Shane and C.R. N. Heginbotham and J. Boenigk. Comparing our results obtained from ligand-binding experiments with those of channel activation [3] reveal that the KD of cAMP-binding coincides with the K1/2 of channel activation by cAMP. The KD values were 73. The KD of cGMP binding is 2 fold lower than K1/2 of cGMP-dependent channel activation. (B) Competition between cAMP (closed circles) or cGMP (open circles) and 8-NBDcAMP (1 µM) for binding to CNBD (1 µM).residue is also a crucial determinant of cNMP-binding to the CNBD of the hyperpolarization-activated and cyclic nucleotidegated (HCN) 2 channel [5]. Kaupp and R. The KD value was 17. A corollary is that the binding event is not affected by intersubunit contact and that binding sites in the tetrameric channel act independently of each other. Kaupp EMBO Rep. 2: Ligand binding to the CNBD protein. L. The CNBD in CNG channels is lacking this particular residue. 8-NBD-cAMP fluorescence in the absence of the CNBD protein was subtracted.3 µM. Rev. Cukkemane.5 nM (cAMP) and 296. Cell 119. E. These findings strongly suggest that binding and gating in the mlCNG channel is a noncooperative process. However. The KD values were 22. Gensch. Olson and E.H.e. These results show that the binding affinity is very similar for the tetrameric channel and the wildtype CNBD.7 µM (cGMP). Zagotta. Morais-Cabral. Physiol. 8. mlCNG represents a cAMPgated channel. i. [4] U. Seifert. 615 (2004). Nimigean. Novak. 124. R. [3] C. The solid line represents nonlinear least-squares fit. Silverman. 203 (2004). [1] A. Nature 425. [5] W. activation of CNG channels expressed in photoreceptors or olfactory neurons is a cooperative process.B. R. B. Several residues in the CNBD as well as at sites distant from the CNBD affect cyclic nucleotide binding [4]. Miller. Olivier. Inset: emission spectrum of 8-NBD-cAMP (1 µM) in the absence (black) and presence of CNBD protein (1 µM) (red). Fig.D. 769 (2002). Interestingly.5 µM) on binding to increasing concentrations of the mutant CNBD (R348A). The KD value was 7. Black. the mutated CNBD has similar binding affinities for cAMP and cGMP. (C) Increase of fluorescence of 8-NBD-cAMP (0. W. the affinity for cAMP-binding is 4 to 5-fold higher than for cGMP-binding suggesting that physiologically.6 nM. T. the molecular basis that determines ligand affinity and ligand selectivity of CNG channels still is not well understood. Physiol. K.5 µM). This 67 .C. J. 200 (2003). Seifert.9 µM (cAMP) and 27. 82.B. Grueter. ~ 400 fold lower than for the wild-type CNBD or mlCNG channel. K. (A) Increase of 8-NBD-cAMP fluorescence on binding to the CNBD protein (0. Gouaux. U. We observed a pronounced change of the binding affinity in the mutated CNBD (R348A).M. These results clearly indicate that both. [2] G.N.9 nM (cGMP). The mutation of this arginine residue results in a CNBD that could be purified in cAMP-free form. B.M. W. The KD values derived for R348A are in the micromolar range. The arginine residue interacts directly with cAMP. Clayton. Gen. This indicates that the arginine residue may be one of the determinants of ligand selectivity and sensitivity. T. In contrast to mlCNG. Young. 749 (2007). bipolar cell densities. Most importantly. respectively.and OFF-ganglion cells.Parallel Information Processing Starts at the First Synapse in the Visual System F. For example. While recording. type 4 bipolar cells show no inward currents and. Each bipolar cell type expresses a unique inventory of ion channels The light response of bipolar cells is shaped by two different means: by the precise timing of excitatory and inhibitory input and by the kinetics of the receptors and ion channels involved in the generation of the input and in the propagation of the electrical signal. The traces show representative current recordings of type 4. Mataruga1. In type 5 bipolar cells. their shape differs from those found in type 5 bipolar cells. In type 6a bipolar cells. Finally. In the “classical” rod pathway. while outward currents are conducted by outwardly rectifying potassium channels. The 68 . These results suggest that bipolar cell types differ in their functional properties. Using the patch-clamp technique in the whole cell configuration we studied the expression of voltage-gated ion channels in bipolar cells in rat retinal slices. however. connectivity to photoreceptors. the membrane potential was clamped to values from -65 to -125 mV in -15 mV increments in a family of 500 ms long hyperpolarizing voltage steps. we extended our studies on the identification of bipolar cell marker proteins. Each cone feeds its signal into every bipolar cell type. Puller2. currents carried by calcium-activated chloride channels are superimposed on HCN channel currents. large sustained outward currents are measured (seen as upward deflection at the end of the trace). and type 6a bipolar cells. At least ten cone bipolar cell types and one rod bipolar cell type provide the direct pathways from the photoreceptors to the ganglion cells that relay the information to the brain.and the OFF-pathway via AII amacrine cells. Wässle2 1 2 ISB-1: Cellular Biophysics MPI Brain Research. For example. Figure 1 shows all bipolar cell types revealed in our study. we identified bipolar cells that provide for the recently proposed “alternative rod pathway”. Note that the axons of different bipolar cell types stratify at different levels of the inner plexiform layer (IPL). Müller1. therefore. the cone pedicle provides output onto at least ten different postsynaptic bipolar cells. rods feed into rod bipolar cells that provide input into both the ON. investigated the connectivity and the functional properties of all bipolar cell types. We. the light response of rod and cone photoreceptors is processed by an elaborate neuronal network (for review see [1]. Bipolar cells establish parallel pathways In the mammalian retina the bipolar cells provide parallel pathways to relay the information from the photoreceptors to the ganglion cells. [2]). type 5. do not express HCN channels. H. these bipolar cell types form the basis for parallel processing of visual signals. C. Upon depolarization to 0 mV. indicating differences in the expression of ion channels. Thus. Ivanova1. these voltage steps elicit pronounced slowly activating inward currents (seen as downward deflections). We found that the dendritic trees and axon terminals of each bipolar cell type tile the retina without much overlap. Each and all cones are connected to at least one member of any given type of cone bipolar cell [3]. From the holding potential of -60 mV. the inward currents are carried by hyperpolarizationactivated and cyclic nucleotide-gated channels (HCN channels). Type 6a bipolar cells show inward currents. Current traces recorded from other bipolar cell types look different. Haverkamp2. Cones feed into ON. therefore. In a highly complex synaptic formation. and bipolar cell axonal tiling in the mouse retina. E. A. In a collaboration with the MPI for Brain Research in Frankfurt. Patch-clamp recordings revealed that each morphologically identified bipolar cell type expresses a unique inventory of voltage-gated ion channels. We found that each morphologically identified bipolar cell type expresses a unique inventory of voltage-gated ion channels suggesting that different bipolar cell types differ in their functional properties. D-60528 Frankfurt In the mammalian retina. each one being specialized to filter certain aspects from the visual information.and OFF-cone bipolar cells that excite ON. S. These membrane currents are conducted by different ion channel types. cells were filled with Lucifer yellow to reveal their morphology. parallel information processing starts at the very first synapse in the visual system. H. Haverkamp. F. Müller and S. M. We performed an immunohistochemical analysis on the level of light and electron microscopy to identify the bipolar cells and ganglion cells that are involved in this “alternative rod pathway” of the mouse retina. Neurosci. 11. Current traces of different cell types show characteristic differences. Rev. mitochondrium. horizontal cell processes. Ivanova and F. indicating differences in ion channel expression. Kremmer and F. Vis. one of which was hitherto unknown. E. Müller. 29. Moreover. J. 69 . 431 (2001). type 5. 71. Bottom: Representative current recordings of type 4. These newly identified cell types represent the basis of a neuronal circuit in the mammalian retina that could provide for an alternative fast rod pathway [5]. 502.B. [4] E. would not have been classified as a distinct cell type [4]. 1123 (2007). Neurol. C. Wässle and B. 143 (2006). Opinion Neurobiol. 447 (1991). Neurosci. The very sensitive rod photoreceptors are suitable for vision at low (scotopic) light levels whereas the less sensitive cones provide for daylight (photopic) and colour vision. We identified two types of OFF-bipolar cells. 106 (2009). Mataruga. putatively postsynaptic OFF-ganglion cell. Boycott. Curr. J. dots. The cones are connected to the cone bipolar cells that synapse onto the ganglion cells. Fig. Wässle. Puller. 2: Electronmicrograph. For a long time it was thought that rod signals are only relayed by one type of bipolar cell to the inner retina. Comp. based on morphological criteria alone. 1: Top: Different bipolar cells in the retina stratify at different levels of the inner plexiform layer (IPL). 23. [2] R. The axon terminals of these bipolar cells co-stratify with the dendrites of a large. our combined electrophysiological and morphological approach allowed us to describe a new type of rat ON-cone bipolar cell (type 6b) that. that form contacts at both cone pedicles and rod spherules. Masland. An alternative pathway for rod photoreceptor signals The retina functions over a wide range of light intensities.different repertoires of currents can serve as “finger prints” to identify the bipolar cell types. Fig. Dendrites of one type of OFF-cone bipolar cell labeled by antibodies against protein kinase A (arrows) form basal contacts at a rod spherule (rs). Müller. Recent evidence suggested an alternative route in which rods directly contact some types of OFF-cone bipolar cells. [1] H. rod bipolar cell dendrites. [5] A. the rod bipolar cell. Physiol. and type 6a bipolar cells (for details see text). [3] H. asterisks. To monitor changes of the Clconcentration during exposure to inflammatory mediators. most likely due to post-translational modification of NKCC1 and KCC2 and thereby change the excitability of DRG neurons. we loaded DRG neurons with the Cl-sensitive fluorescent dye MQAE. The MQAE fluorescence is quenched by Cl.Maturation and Modulation of Neuronal Chloride Homeostasis T. Excitatory Cl. Heidelberg. In most neurons of the adult CNS. Whether the opening of Cl. A. leading to channels causes Cl depolarization and excitation of the cell.e. D.and is well suited to monitor time-dependent changes of the intracellular Cl level. and in neurons challenged by ischemia. Warm colours represent high intracellular Cl (small τ values). Regulatory mechanisms that control the activity of Cl.currents occur in neurons which accumulate Cl . that DRG neurons undergo a developmental transition of chloride homeostasis during the first three postnatal weeks. In a second approach. This effect coincided with 70 . or neurological disorders. Cluptake is mainly mediated by NKCC1 [3] activity.or glycine-activated ionotropic receptors mediate Cl. Frings2. - The data indicate that inflammatory mediators raise intracellular [Cl-] significantly within 2 hr of treatment with inflammatory mediators. Neuronal activity can be strongly influenced by Clcurrents.concentration. 1). inflammation. This may be the basis for increased pain perception during inflammation.import and export can have profound effects on the Nernst-potential for chloride ECl and thereby determine whether the opening of Clefflux. DRG neurons display a heterogeneous pattern of Cl concentration. Gilbert2. the internal chloride concentration [Cl-] is low.g. Univ.effects is determined by Cluptake and Cl extrusion pathways in the cell.influx. in immature neurons of the CNS. Notably. is controlled by the membrane potential and by the intracellular Cl concentration. (B) Quantitative analysis of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after starting the treatment with inflammatory mediators. i. e. K.ions is controlled by KCC2 [4]. The balance between inhibitory and excitatory Cl. Woitecki2. C. Changing the balance between Cl. S. Gensch1. whereas KCC2 couples Cl extrusion to K+ efflux. We have studied the effect of inflammatory mediators (i. Funk2. Our results show. opening of GABA. Hence. The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Cl concentration. We have determined the intracellular Cl concentration [Cl ] of somatosensory neurons using twophoton fluorescence-lifetime imaging microscopy (2P-FLIM) with the Cl--sensitive dye MQAE [1] and monitored the expression of NKCC1 and KCC2. whereas the efflux of Cl. Möhrlen2 1 2 ISB-1: Cellular Biophysics Institute of Zoology. CGRP or substance P) on the [Cl ] as well as on the expression of NKCC1 and KCC2 in DRG neurons. The recorded fluorescence lifetime was colour-coded for microcopic images such that warmer colours represent higher Clconcentrations (Fig. 1: Monitoring intracellular Cl concentration in DRG neurons.transporters include phosphorylation as well as dimerization.e. we studied the effects of inflammatory mediators on Cl homeostasis in dorsal root ganglion (DRG) neurons [2]. lead to inhibition of the cell. in olfactory sensory neurons. Franjic-Würtz2.e. i. Inflammatory mediators raise the intracellular Cl. As an experimental parameter we analyzed the fluorescence lifetime of MQAE. which is independent of the intracellular dye concentration. + + A Na -K -2Cl co-transporter NKCC1 provides the main route for Cl uptake. F. D-69120 Heidelberg influx that causes hyperpolarization and inhibition of the neuron.selective ion channels leads to excitatory or inhibitory currents. or to Cl- Fig. the 2P-FLIM measurements revealed that virtually all DRG neurons visible in the image increase their [Cl ] in response to the inflammatory stimulus. (A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with MQAE. Cation-coupled Cl cotransporters control the [Cl-] in neurons. substance P and calcitonin-gene related peptide (CGRP). In contrast to CNS neurons. Funk. This notion is supported by our results on DRG neurons treated with inflammatory mediators in which Claccumulation coincides with phosphorylation of the NKCC1 transporter. Dev.1. Frings. we investigated the expression level of several Clcotransporters. in a reatehr heterogeneous distribution of Cl. K. Rather than on changes of the expression level of electroneutral cation-chloride cotransporters. Somatosensory neurons in the dorsal root ganglia undergo a transition of Cl. [1] D. 32 (2008). 2 with 2PFLIM measurements of MQAE. we did not detect changes in the transcript level during maturation in any of these genes. K. G. 25. that of KCC2 declined after 3 hours of treatment. T. somatosensory neurons maintain a heterogeneous pattern of Cl values. C. [3] B. Int. J. Berglund. Woitecki. the main neuronal Cl. Augustine and R. Jentsch. τ.homeostasis in somatosensory neurons develops during postnatal maturation into a state where the [Cl ] can be regulated individually in each neuron. C.levels of the early postnatal days. Newborn neurons show almost uniformly high [Cl ] (≥70 mM). 2: Determination of Cl in somatosensory neurons by 2P-FLIM.levels.importer and exporter.accumulation observed by 2P-FLIM. respectively. Immunological staining of DRG sections with antibodies specifically binding to phosphorylated NKCC1 showed an increase of 18% and 45% of the phosphorylated form of NKCC1 after 1 and 2 hrs of inflammatory treatment. Pond. T. - 71 . Schwartz-Bloom.homeostasis after birth (Fig. within postnatal weeks 1 – 3. Kuner. Gensch. In a second study we examined the maturation of Cl. Taken together. J. This results. Neurol. the efficiency of Cl accumulation sets the level of [Cl ] and.J. 468. Gilbert. Our studies demonstrate that Cl. exposed an inverse regulation by the inflammatory mediators. most somatosensory neurons decrease their [Cl ] to some extent. Immunohistochemistry of NKCC1 and KCC2. Mol. Möhrlen. Somatosensory neurons have a high internal [Cl ] after birth (Fig. Surprisingly. Therefore. [2] K. Neurosci. hence.enhanced phosphorylation of NKCC1. Gensch. To unravel the molecular basis for the maturationdependent transition of the Cl concentration. These results suggest that phosphorylation of NKCC1 in DRG neurons causes the early (< 3 hr) phase of enhanced Cl. is colourcoded as in Fig. 479 (2007). [4] V. whereas the majority of somatosensory neurons contain either intermediate or low levels of Cl-. This process parallels the developmental “chloride switch” in the CNS. Feng.homeostasis during the first three weeks of postnatal development. Möhrlen and S. Comp. However. roughly a third of the cells maintain the high Cl. However. (c) 2P-FLIM images illustrating [Cl ] levels in somatosensory neurons from newborn (P1-P4) and adult rd (≥3 week) mice. 2). this regulation may be achieved by postranslational modification of existing proteins. while most CNS neurons achieve homogeneously low Cl. as displayed in Fig.H. Stein. In the 2P-FLIM image. 57 (2004). S. 1396 (2006). Neurosci. T. While the NKCC1 immunosignal increased. Hermanns-Borgmeyer and T. A. 2). Fig. The colour scale illustrates the false-colour representation of [Cl ] in the following images. During maturation. it is highly unlikely that transcriptional regulation underlies the maturational change in Cl homeostasis in somatosensory neurons.D.B. the fluorescence lifetime. (b) Calibration of 2P-FLIM signals in isolated DRG neurons with [Cl ] set to the indicated values using ionophores. (a) Comparison of fluorescence intensity (left) and lifetime (right) images from the same DRG. Frings and F. G.levels in a DRG. J. 26. Franjic-Würtz. F. resulting in a heterogeneous mosaic of [Cl ] levels in the ganglia of mature animals. may control the sensitivity of adult sensory neurons. I. Franjic-Würtz. Funk. Pain 4. 72 . crystallized and high-resolution X-ray structures are determined from ground and intermediate states of their working cycles to obtain a nearly complete time-resolved image of their mechanisms. Georg Büldt One ultimate goal in biophysics is to understand the functions of proteins and macromolecular complexes as well as whole cellular processes like signal transduction pathways in their cellular environment.ISB-2: Molecular Biophysics Director: Prof. Protein unfolding and refolding as well as the interactions between proteins are studied in aqueous solution and in membranes. For most biophysical techniques it is difficult or in many cases impossible to apply physical methods to living cells. ISB-2 follows several approaches to achieve this goal: • Proteins or complexes are isolated. • • 73 . Cell-free transcription and translation systems in combination with fluorescence single molecule spectroscopy allow us to accompany protein synthesis and folding at the ribosomal machinery. represents a helical bundle comprising three TPR motifs. Finally. Members of the FKBP family have also been identified in plants. Weiergräber1 . which has been implicated in plant development and response to environmental stress. connected to one or more FKBP domains. Another feature shared by many FKBPs is the ability to act as peptidylprolyl cis-trans isomerases (PPIases). The X-ray structure of the former construct reveals a canonical FKBP-type fold. We have determined the X-ray structure of the soluble portion (aa 1-339) of FKBP42. In addition to a single FKBP-type domain. FIG. Intriguingly. whereas the TPR domain appears to be responsible for functional association with vacuolar transporters MRP1 and MRP2. Like many other multi-domain immunophilins. have also been termed "immunophilins". 1: of PPIase activity and does not display measurable affinity for FK506. consisting of a five-stranded antiparallel β -sheet wrapped around a short α-helix. We propose that FKBP42 may enhance auxin transporter activity by stabilizing a conformation of the nucleotide binding domain which is competent for ATP loading and/or hydrolysis. PGP1 and PGP19 have been shown to directly mediate cellular auxin efflux. respectively [3]. The N-terminal domain displays an FKBP-type fold belonging to the αβ -class. 74 . Recently. typically a tetratricopeptide repeat (TPR) domain. J. it contains a tripartite TPR motif and a hydrophobic C-terminal membrane anchor [1].FKBP42. an immunophilin modulating ABC transporter activity O. These effects are mediated by direct interaction with auxin carriers belonging to the ABC transporter superfamily. we predict that the interaction of FKBP42 with the heat shock protein HSP90 will occur in a similar way as found for other immunophilins. we have built a model for the complex of FKBP42 with the Cterminal nucleotide binding domain of the auxin transporter PGP1. We have crystallized fragments of FKBP42 covering the N-terminal domain (aa 1–180) and the entire soluble portion (aa 1–339). together with the family of cyclophilins. the protein interacts with HSP90. Based on sequence conservation. Mean B factors are indicated by a colour gradient from white (B < 35 Å2 ) to red (B > 90 Å2 ). The C-terminal segment. The FK506 binding proteins (FKBPs) represent a ubiquitous protein family named after the role of several members as primary targets of FK506-type immunosuppressants in animal and human cells. indole 3-acetic acid. FKBP42 is devoid Ribbon representation of the FKBP42 structure. Side chains establishing inter-domain contacts are indicated. the FKBPs. also termed TWISTED DWARF1 (TWD1) due to the reduced height and disoriented growth of null mutants. Multi-domain FKBPs are characterized by additional protein modules. Batra-Safferling1 . and this activity is regulated by FKBP42 [2]. Based on this activity. The predominant auxin. The FKBP domain of FKBP42 has been demonstrated to physically interact with plasma membrane-localized ABC transporters PGP1 and PGP19. is synthesized at the shoot apex and undergoes a basipetal transport which is crucial for the establishment of plant polarity. is a type-II membrane protein comprising 365 amino acids. H. R. FKBP42 from Arabidopsis thaliana. Phytohormones of the auxin family represent essential regulators of plant growth and development. which implicates these proteins in peptide folding and chaperoning processes. Granzin1 1 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) FKBP42 is a type-II membrane protein which plays an important role in the regulation of polar auxin transport in higher plants. on the other hand. I. O. Murphy. 3: Model of the FKBP42-PGP1 complex. To this end. We are currently striving for experimental validation of our models by obtaining X-ray structures of FKBP42-ABC transporter complexes. 251–255 (2006). A. J. The crystal structure of the larger construct is shown in Figure 1. Büldt. respectively. aureus. the C-terminal portions of PGP1 and PGP19 containing the second nucleotide-binding domain (NBD2) bind to the FKBP fold. S. Biol. Experimental evidence indicates that regulation of ABC transporters by members of the FKBP family may not be restricted to plant cells. Schulz and J. Lee. To visualize the overall architecture of the complexes and the orientation of the components with respect to the membrane. Schulz and M. T. FKBP42 is unique among large immunophilins in that both the FKBP and TPR domains have been shown to interact with ABC transporters in vivo. investigation of a fulllength ABC transporter. we propose that the interaction of HSP90 family members with FKBP42 is mediated by the invariant Cterminal pentapeptide MEEVD engaging a TPR domain of the binding partner via a "two-carboxylate clamp" mechanism. Eckhoff and O. may ultimately be feasible. R. 263–276 (2002). Kamphausen. whereas the equivalent domains in MRP1 and MRP2 associate with the TPR module. FEBS Lett. bottom view. Our models featuring the complexes of FKBP42 with PGP1 and MRP1 may serve as working hypotheses stimulating further investigation of these novel protein-protein interactions. K. These include the association with multi-domain immunophilins. Weiergräber.FIG. FIG. Granzin. Granzin. J. their arrangement is unique and may have important functional implications. 364. Weiergräber. U. 2: Model of the FKBP42-PGP1 complex. side view. 30603–30612 (2006). Vincenzetti. H. Bailly. G. The N-terminal part of the molecule (left) displays the expected FKBP-type fold. either alone or as fusion constructs with their respective N-terminal counterparts. Plant J. Geisler. Based on sequence conservation. Rahfeld. B. Bouchard. In the context of our current efforts towards improving crystallization of membrane proteins. 75 . 799–809 (2006). represents a helical bundle (right). The crystal structure of FKBP42 described here represents the first three-dimensional structure of a multi-domain immunophilin from plants. 363– 365 (2005). Blakeslee. [4] O. H. A. as anticipated from the early sequence-based classification as a TPR domain. Fanghänel. [2] R. J. the NBD2 model (blue) has been incorporated into the crystal structure of Sav1866 from S. [1] T. we have developed homology models for the NBD2 of PGP1 and MRP1 representing the two classes of ABC transporters considered here. which had originally been investigated in animal systems. Neumann. The domain interface of FKBP42 is composed of a hydrophobic network surrounded by hydrogen bonds and electrostatic contacts [5]. V. Acta Cryst. 580. are conserved in plants. on the other hand. Kamphausen. Eckhoff and J. [5] J. The absence of detectable PPIase activity as well as FK506 affinity could be explained by the active site being partly occluded by protein side chains [4]. Paponov. Oehring. In order to improve our understanding of these novel protein-protein interactions. 281. The C-terminal segment. Palme. H. The resulting models were subjected to an in silico docking procedure together with the appropriate domains of our FKBP42 structure. F61. Chem. B. Mancuso. [3] A. Eckhoff. we are establishing expression and purification of the C-terminal nucleotide-binding domains of PGP1 and PGP19. J. J. S. A. D. 32. Schulz and O. B. A. with or without an associated FKBP42. Figures 2 and 3 present an updated model illustrating the interaction of FKBP42 with PGP1 (unpublished data). J. Specifically. Mol. C. S. While the overall architecture of the two domains matches the known characteristics of the FKBP and TPR folds. Granzin. Weiergräber. Recent evidence has indicated that many of the protein-protein interactions in the HSP90 chaperone complex. Biol. it is expected that the presence of one dye molecule per arrestin in the concave regions as well as in the area of the helix should be sufficient to exert a clear negative influence on binding efficiency to P-Rho*. By simultaneously modifying both domains with one Alexa 633 moeity the binding capacity was reduced.Granzin1 1 2 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Institute of Structural Biology and Biophysics: Structural Biochemistry (ISB-3) Arrestins play a crucial role in regulation of signal transduction of G protein-coupled receptors (GPCRs) [1]. 2009). Dasara Raju1. Schlesinger1. odorants and neurotransmitters to the interior of cells via activation of their cognate G proteins. In 1998 we published the first 3-dimensional X-ray structure of arrestin-1 from bovine rod outer segments in the receptor-free form [2]. the structure of arrestin in complex with the activated and phosphorylated GPCR remains elusive. B. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. G. Our light scattering experiments suggested that two rhodopsin molecules bind to the two domeshaped structural motifs of arrestin (Figure 1) [3]. R. We have used several complementary methods to address local aspects of the arrestinrhodopsin interaction. GPCR signaling is shut off by a conserved two step process. These findings are not consistent with the current working model of arrestin binding to rhodopsin (see literature). Arrestins regulate a large number of different GPCRs and serve additional functions beyond desensitization of GPCR signaling [1]. Granzin unpublished ) and rhodopsin (Protein Data Bank ID: 1U19) interaction. König2.e. The overall fold of the basal state of arrestin is conserved across the various arrestins (see above). High resolution liquid state NMR can determine the structure and orientation of peptides in the rhodopsin-bound state (paper submitted. i. This observation indicates that both concave sites participate in binding. However. The docking has been carried out manually without energy minimization and the distance between the receptor monomers is adjusted based on the work of Fotiadis et al. 76 . FIG. J. Until now it is unclear how arrestin interacts with rhodopsin. This would also be expected when considering earlier investigations which implicate a large number of residues in these regions. Batra-Safferling1. phosphorylation of the GPCR followed by tight binding of arrestin. GPCRs convey a large variety of extracellular stimuli including light. In this case. J.Visual signal transduction studied by a variety of biophysical methods R. [5]. G. Büldt1. which requires the accessibility to different regions that are responsible for the binding and recognition process of P-Rho*. 1: A possible model of arrestin (residues 6-388. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores. Four types of arrestin can be distinguished in vertebrates: rod arrestin (arrestin-1) and cone arrestin (arrestin-4) are restricted to the corresponding compartments of the retina while non-visual β-arrestin-1 (arrestin-2) and β-arrestin-2 (arrestin-3) are widely distributed in various tissues. A. 127-128 (2003). Fotiadis. Furthermore crystallization of the complex (arrestin-rhodopsin) is in preparation. G. D. Chen. Krafft. [2] J. USA 104.V. Büldt. In addition we will perform timeresolved fluorescence imaging experiments to visualize the interaction of arrestin with activated rhodopsin (in collaboration with U.J. Natl.and C-terminal domains of arrestin are depicted in dark blue and green. [1] K. B. Residues 67 to 77 in the unstructured loop connecting β-strands V and VI are colored in yellow. Filipek. Nature 391. Acad. Liang. 83. FU Berlin). Granzin. We speculate that the largely αhelical conformation of the Meta II-bound peptide reflects the conformation of the corresponding “finger loop” region in the arrestin-P-Rho* complex (Figure 2a. Skegro. Proc.Sci.K. McClatchy. Palczewski. Lefkowitz. Y. FIG. A.K. 25. U. Alexiev. Granzin. Sci. Saperstein. Choe. 918-921 (1998). G. [3] D. A. Büldt.P. These binding studies indicate that Arr(67-77) competes with arrestin for its binding site on Meta II-rhodopsin. S. Photochem. Gurevich. Engel and K. Trends [5] D. Pharmacol. J. Schlesinger. K. Krafft. J. J. D. Loop residues 67-77 have been replaced by the NMR structure of Meta II-bound peptide Arr(67-77) followed by energy minimization.R. Shukla. These observations support the sequential multi-site binding model of receptor-arrestin interaction proposed by Gurevich and Gurevich [4] and suggest a crucial role of arrerstin loop V-VI in the recognition of rhodopsin activation. Receptor binding of Arr(67-77) strictly requires rhodopsin activation but is rather insensitive to receptor phosphorylation. R. Wilden. A. [4] V.V. Gurevich and E. Xiao. the N. S. Y. Labahn. Photobiol. Hofmann. M. Shenoy. Pulvermüller.FIG. 12011-12016 (2007). 2b: Arrestin loop V-VI adopts an α-helical structure (boxed) upon binding Meta II. Yates. Nature 421. 385-393 (2007). 105-111 (2004). H. 77 . 2a: Crystal structure of uncomplexed arrestin shown as a ribbon representation. and R. b). Zhao.-W. respectively.B. B. We have started time-resolved fluorescence anisotropy measurements which will give further evidence for the mode of interaction between the two molecules. The crystallographic symmetry axis is located between TM1–TM2 and TM1’–TM2’ . α-Helices are in red for the receptor and green for the transducer. Homologs of sensory pathway in which the complex is involved occur in all the three kingdoms of life.Signal transduction by sensory rhodopsin II-transducer complex V. TM2’ (where a prime indicates the right-hand complex. The expected dimer of the complex is formed by a crystallographic two-fold rotation axis. We have obtained X-ray structures of the ground state and photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. A notable exception is found for Tyr 199. The asymmetric unit contains one complex. as 78 . The F–G loop region fixes the transducer by several contacts as well as by three hydrogen bonds between Thr 189 (SRII). TM1’ . Glu 43 (TM1) and Ser 62 (TM2). Unfortunately. The data showing a signal transfer from receptor to transducer indicate that HtrII-114 forms a functional complex with its cognate receptor SRII. β-strands are in blue and coils in grey. A second anchor point is observed in the middle of the membrane where. The same was true for the case of the NpSRII/NpHtrlI complex.8 Å. The established mechanism might also be relevant for eubacterial chemoreceptor signaling. Crystallization of the receptor–transducer complex. However. which constitute a family of seven-helix retinal membrane proteins. a member of the two-component signaling cascade. a signal transduction chain. Labahn1. most notably in enteric bacteria in which the chemotaxis has been extensively studied. G. 1). J. Archaea and Eukaryota. the binding of the transducer to helices F and G hardly interferes with the side-chain arrangement of the receptor. The labels of the symmetry related complex are marked by a prime. 3]. After excitation by blue-green light NpSRII triggers. Fig. The transmembrane helices F and G of the receptor are in contact with the helices of the transducer [2. are distributed throughout the Bacteria. Membrane receptors play key roles in different cell functions. M. by means of a tightly bound transducer protein (NpHtrII). Gordeliy1. which is located in the middle of four transmembrane helices: TM1. Büldt1 1 2 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Max-Planck-Institute for Molecular Phisiology Microbial rhodopsins. there is no molecular picture of the signal transduction mechanism for any receptor. The microbial phototaxis receptor sensory rhodopsin II (NpSRII) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behavior of the bacterium. Engelhard2. Obviously. TM2. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes.8 Å).1: Ribbon diagram of the top view from the cytoplasmic side. the atomic resolution structure of the complex as well as molecular events leading to signal transduction were still missing. The results provided a first atomic model for signal transfer within the membrane part of the receptor. Our goal was to obtain such information via elucidation of high resolution structure of the complex and its intermediates. The aromatic plane of Tyr 199 has turned in the complex by about 90˚ and is now pointing into the direction of TM2 where its phenolic group forms a hydrogen bond to Nδ(2)Asn 74 (2. has been achieved successfully using a shortened transducer comprising the two transmembrane helices (TM1 and TM2). Thin orange crystals of SRII in complex with HtrII114 grown in lipidic cubic phase displayed an orthorhombic shape of about 140 μm in size and diffracted to 1. FIG. Electron paramagnetic resonance data led to the conclusion that the complex functions as a dimer [1]. Recent structural and functional studies on the sensory rhodopsinII/transducer complex have yielded new insights into the mechanisms of signal transfer across the membrane. and Navarro J (2001) Proc.. et al. thereby enabling proton transfer. Difference densities clearly show changes in the position of the main chain that are not present in the K state (for example Arg 72) (Fig 2).mentioned above. Pebay-Peyroula E.. Sci.9 Å to 2. During the transition to the signaling state (late M) these disturbances increase so that in late M fewer hydrogen bonds connect helices C and G. light-activated signal transfer from receptor to transducer originates in the isomerization of retinal. The main effect on TM2 is a clockwise rotation of about 15˚ and a tilt of the helix.. 2: Changes in the structure of helices C and G of ground state (red) and late M state (yellow) including water molecules as red and yellow balls.7 Å as a result of the movement of the Schiff base. The hydrogen bond structure (Asp 201-Oδ· ·W2· ·W4· ·Arg 72Nε) is retained.L. Edman K. Both causes give rise to changes in the tertiary structure of the receptor in late M. The isomerization of the retinal bound to Lys 205 gives rise to a displacement of its Cε and Cδ atoms by 1.. Engelhard M.1 Å. The crystals of the complex were illuminated to freeze-trap the K and M intermediates. Therefore the pentagonal structure of hydrogen bonds including water molecules W1 to W3 and the oxygen atoms of the aspartic residues Asp 75 and Asp 201 found for the ground state can no longer exist in the K state. [1] Hoff W. Natl. In the reaction path from the ground state through K to late M the temperature factor of Asp 75-Oδ2 varies from 18 Å2 (BG) through 31 Å2 (BK) to 22 Å2 (BM) and the distance between Asp 75-Oδ2 and the Schiff-base nitrogen relaxes from 4. The ground-state complex is shown in red and the late M-state complex in yellow FIG. but also for other dimeric receptors. (2001) Cur.. et al. Acad. which produces a redistribution of charges. Gordeliy V.Biophys. 5. The charge separation between the protonated Schiff-base nitrogen and Asp 75-Oδ increases from 4. The distance from Asp 201-Oδ to the Schiff-base nitrogen is reduced from 4. 797-799. (1997) Annu. the phenolic hydroxyl of Tyr 199 bridges to Asn 74..USA 98. which induces local changes in the hydrogen-bonding network in a region near the retinal (K state). Consequently these helices now have more freedom to move. when proton transfer has occurred. Efremov R. I.9 Å in the K state to 4. FIG. Nature 440.3 Å in late M. Büldt G..223-258 Gordeliy V. This shift reduces the distance between Lys 205-Cε and the ground state position of water molecule 1 (W1-O) from 3. Landau E. Biomol. New information is of significance not only for bacterial phototaxis and chemotaxis [5]. Jung K. In the K state structural changes with respect to the ground state are observed within a sphere of 9 Å diameter around the central water cluster on the extracellular side of the retinal [4].5 Å in the K state.. Biol. with the hinge at Ser 62.9 Å in the K state.. although W2 and W4 shift towards the retinal by about 1. Labahn J.D...-H. Struct. Biology 9. Moukhametzyanov R. Engelhard M.. 723-724.10131-10136.9 Å at the cytoplasmic side (Leu 77). 3: Schematic picture of helical displacements viewed from the cytoplasmic surface. 484–-487 (2002).. Rev. Büldt G. Neutze R..5 Å. During the transition from K state to M1 state the proton of the Schiff base translocates to Asp 75.3 to 2. Nollert P. that amounts to 0. 115-119 (2006) Spudich J. W1 is driven away from its original location [2]. (2002) Nature Str. and Spudich J. Royant A. Nature 419. Opinion in Chem. Signal transfer to the transducer takes place within the interface of receptor helices F and G and transducer helix TM2. and for the first time might lead finally to a general model of transmembrane signal transduction [6]. Thus. [2] [3] [4] [5] [6] 79 . 26. Klare J. Newman R.2 Å to 4.M. In late M state a further hydrogen bond connecting helices C and G in the K state through water molecules W2’ and W4’ has vanished. These alterations in the hydrogen-bond network change the pKa values of the Schiff base and Asp 75. The bilayer is locally 2-dimensional like a cell membrane and therefore allows the incorporation of membrane proteins. Labahn2 1 2 Qiagen Research&Development. It allows fast screening of crystallization conditions employing automated liquid handlers suited for the 96 well crystallization format.2 mg monoolein and 350 nl H2O at 22°C. The in-mesophase crystallization [1] unfortunately requires cumbersome manual work like weighting mg-quantities for every single crystallization experiment. The cubic phase Pn3m consists of a bi-continuous bilayer that separates two channel systems of aqueous phase. FIG. typically when the concentration of the precipitating agent in the droplet is approximately the same as in the reservoir (Fig. Schäfer1. The kinetics of the self-organisation of monoolein can be observed by incubating solid monoolein with water. This end point of the experiment is to be refined experimentally to reach a droplet composition sufficiently supersaturated to force nucleation and subsequent crystallization of the protein. Hilden Institute of structural Biology and Biophysics: Molecular Biophysics (ISB-2) The presented method to crystallize membrane proteins combines the advantages of the mesophase crystallization method and the classical vapour diffusion crystallization method. In vapour diffusion experiments with monoolein the dehydration can be experimentally realized by enclosing the wetted monoolein together with a reservoir solution which takes up water from the gas phase that separates the two condensed phases (Fig. In vapour diffusion type crystallization experiments typically a protein solution and a solution containing a precipitating agent are mixed. J. The formation of the optically isotropic cubic phases or birefringent lamellar phase can be observed with a polarization-microscope.Vapour diffusion for in-mesophase high throughput crystallization J.3). This is detrimental to high-throughput screening procedures using automated liquid handling systems as employed successfully for the crystallization of soluble proteins.. The small number of known membrane protein structures can be directly traced back to problems in obtaining membrane protein crystals for structural investigations. Kubicek1. This can be achieved by adding solid salt [1]. For less than hundred membrane proteins their 3dimensional structure at atomic resolution had been determined though one-third of the proteins inside a cell belong to this class. intransparent monoolein phase transforms within 25 minutes almost completely to the optically isotropic cubic phase. The methods developed for crystallization of soluble protein are inefficient for membrane proteins.1). G. left: broken arrow). But it extends continuously through space and supports therefore diffusion of the protein in three dimensions and crystallization upon dehydration. Experiments with bacteriorhodopsin proof that by this approach crystals of high quality can be obtained. Landau and Rosenbusch [1] used lipidic mesophases to accommodate the specific needs of membrane proteins in a way compatible with crystallization: The lipidic component monoolein self-organizes with water into mesophases [2] (Fig. F. whereas the birefringent lamellar phase disappears.2: Swelling experiment with 0. The solid. 80 . Büldt2. Generally the vapour pressure of water above these two phases will be different: Water will transfer via the vapour phase into the reservoir solution until the two condensed phases are in equilibrium.3. This state of affairs is especially bothersome as the natural entry points to a cell are membrane proteins and their assemblies: The lacking knowledge of membrane protein structures translates directly into lacking knowledge of biomedical targets and mechanisms. Dehydration of the mesophase can be achieved by lowering the level of humidity. A small droplet of this mixture will generally be not in equilibrium with undiluted reservoir solution.2). Within an hour ithe formation of cubic phase is observed (Fig. 1: Isotherm from monooolein/water phase diagram. 0 min 1 min 3 min 7 min 25 min FIG. F.1 mm and diffract to 1. 321.3. J.M. cubic phase to lamellar phase (Fig. 21. ID14-1 at a wavelength of 0.8 Å. Kubicek.5). right) the protein containing cubic phase will be dehydrated by the reservoir solution and form lamellar phase (Fig. Natl.3. Rosenbusch Proc. Cupp-Vickery.Crystals of bacteriorhodopsin grown in mesophase using vapour diffusion reach a size of 0. middle) is observed.P. 14532 (1996) [2] H. Sci. Labahn. USA. 1. Experiments with the light-driven bacterial proton pump bacteriorhodopsin from Halobacterium salinarum show that crystallisation occurs upon transformation of the transparent. respectively mol-fraction of protein in the respective phases FIG. Therefore an increasing amount of the protein will be incorporated into the mesophase according to the Nernst partition law: K = Caqueous/Xmesophase C. V.5 Å When solid monoolein is brought into contact with an aqueous protein droplet it will incorporate protein and take up water. The loss of water from the droplet towards the reservoir (blue arrow) enhances the incorporation of protein into the cubic phase. 223 (2000) [3] B. Measured at ERSF.Biol. J. Caffrey Biomaterials. Over days the formation of optically isotropic cubic phase (light grey. After the fast take-up of water by monoolein the slower loss of water in the droplet by vapour diffusion will further increase the concentration of protein in the aqueous phase (Fig. The main parameters of this crystallisation approach [4] that are to be refined experimentally for each type of precipitating agent and for each protein are: • The ratio of protein and monoolein mass • The mixing ratio of volumes in the initial drop • The total initial drop size [1] E.X: concentration. M.Mol. Nucleation of protein crystallization is thought to be brought-by by the local transformation of cubic phase to lamellar phase. G. isotropic. Landau. Grenoble. 3: Hanging drop vapour diffusion experiment with monoolein: The incubation of monoolein (dark grey) with an aqueous droplet mixed from membrane protein (green) and reservoir (red) solution leads to uptake (arrows) of water and membrane protein and subsequent phase transformation within hours. 81 . Schobert. Qiu.43 Å [3] with other crystallisation methods. Büldt Patent pending (2009) FIG.4: Crystallization experiment with Bacteriorhodopsin in monoolein mesophase employing vapour diffusion. Acad. Schäfer. that is considerably further than the best results obtained so far 1.4). 93.2: right to left).14 Å (Fig. Inset shows observed resolution limit at 1.25 mm * 0. S.5: Diffraction experiment with bacteriorhodopsin crystal from in mesophase vapour diffusion. Over weeks to month (right) the cubic phase reverts back to lamellar phase due to dehydration by vapour diffusion. 715 (2002) [4] J. middle). Hornak.14 Å Lately (Fig. J. Smith J. Lanyi J. FIG. Formation of the fluorescent chromophore is a rather slow posttranslational autocatalytic process and the maturation kinetics as well as the folding efficiency between GFP wild type and several mutants differ significantly [3]. Büldt1 1 2 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) RiNA GmbH. We have chosen the GFP Emerald (GFPem) mutant which is characterized by a high folding efficiency and by fast folding and maturation kinetics. one major goal is to understand how polypeptide chain elongation and folding are coupled. such as polypeptide synthesis and protein folding. and to the yields. By the use of a streptavidin-biotin binding assay fluorescently labeled ribosomes were linked to the surface via biotinylated ribosomal protein L4 (displayed molecules are not on scale). The aminofunctionalized cover slide is coated with a layer of PEG which is biotinylated at low concentration. Fluorescence of cotranslational folded proteins was observed from mature fluorescent GFP molecules which carry 31 additional amino acids at the C terminus remaining linked to the ribosome. GFP synthesis at surface-immobilized fluorescently labeled ribosomes was accomplished by using a fractionated cell-free transcriptiontranslation E. Choli-Papadopoulou4. Katranidis1. Our images indicate that approximately 10 – 15% of all visible ribosomes produce a bound mature and fluorescent GFP. Significant differences have been observed between both types of folding with respect to folding rates. to the appearance of folding intermediates. K. J. The sequence of GFPem was elongated by a sequence of 31 amino acids at the C-terminus (spanning the full tunnel length) in order to ensure proper folding of the full length protein outside the tunnel.4. coli system (Figure 1). T. Numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins [1]. Therefore. R. Fitter1. Berlin 3 Max-Planck-Institute for Molecular Genetics. In particular single-molecule studies can yield valuable information about these rather asynchronous processes. Processes precedent to chromophore formation. For imaging fluorescently labeled ribosomes (with ATTO 655) and emerging GFP molecules we employed a dual color fluorescence wide-field microscope (Figure 2). The fastest GFP molecules appeared already within one minute. Greece We demonstrate here by single molecule experiments that a green fluorescence protein (GFP) is produced with a characteristic time of five minutes. Suppression of protein release after synthesis keeps the synthesized GFP bound to the ribosome and allows to image GFP fluorescence for extended observation times. In a next series of measurements we monitored the appearance of individual synthesized GFP molecules as a function of time (Figure 3). 1: Schematic view of surface-tethered ribosomes (only the 50 S subunit is shown).In vitro co-translational folding A. G. In this study we observed green fluorescent proteins (GFP) at a single molecule level after de novo synthesis and folding [2]. It has been demonstrated in several cases that protein folding takes place already during the elongation of the nascent chain (co-translational folding). Schlesinger1. For this purpose surface-immobilized ribosomes were incubated with a reaction buffer within a closed 82 . are fast and last not longer than one minute. In classical folding studies formerly folded proteins need to be transferred into an unfolded state before the (re-)folding process can be studied. Berlin 4 Aristotle University of Thessaloniki. Cell-free synthesized GFPem becomes mature while linked to the ribosome. Geritts2. Nierhaus3. M. Thus it was possible to co-localize fluorescence from labeled ribosomes and from GFP molecules. In our approach a two color single molecule sensitive fluorescence wide-field microscope is employed in order to visualize surface tethered labeled ribosomes and de novo synthesized GFP molecules in real time. FIG. Therefore we conclude that polypeptide synthesis and protein folding together must be faster than one minute. while the corresponding in vivo rate is 10-20 residues per second. K. Fitter. Typical maturation times of other GFP mutants of the S65T type range from 15 to 45 minutes. I. which is one of the fastest maturation times for a GFP mutant observed so far. Therefore a characteristic time obtained from the kinetic data is related to the succession of all sub-processes. Büldt and J. 3: As an example integrated peak intensities are shown as a function of time for fluorescent GFP molecules appearing at different times after the initiation of biosynthesis. 2009. 759-781.O. Angewandte Chemie Int. To our surprise GFPem fluorescence shows up rather fast with a significant fraction within five minutes after initiating polypeptide synthesis. 272. However. Sample Ar+ Laser 488 nm Microscope objective Beam splitter chromophore formation time requires at least 5-10 minutes for de novo synthesized GFP molecules. For our GFP construct with 306 residues (36aa+GFPem+31aa) this would last one to five minutes for a cell free system. D.3 minutes) is related to the chromophore formation. 2: Scheme of the two-color wide field setup used to image fluorescently labeled ribosomes and emerging GFP molecules at the same time with single molecule sensitivity. J Biol Chem. we have to assume that the characteristic time constant obtained from our data (5. Fluorescence of individual GFP molecules can only be detected for a few consecutive exposures before photo-bleaching occurs. Fedorov. Edit. as part of the whole biosynthesis..H. Gregor. Baldwin. Therefore. Rev. [1] A. T. 1997. High rates of folding and maturation are assumed to play a crucial role to reduce unwanted side reactions. G. in our study GFP molecules become detectable only after chomophore formation. we have to account for the polypeptide elongation with a synthesis rate of about 1-5 amino acid residues per second in cell free systems.imaging chamber and after a dead time of 40 seconds a sequence of images was taken every 15 seconds.H. Gerrits. namely polypeptide synthesis. Atta. The corresponding times range from around 4-5 minutes for concentrated proteins in solution to a few ten seconds in single molecule studies or in chaperonin-mediated refolding. protein folding. (iii) Since the characteristic FIG. Katranidis. The corresponding characteristic time constant for the observed process is 5. and thereby improve the efficiency of protein biosynthesis in the cell. Choli-Papadopoulou. An interpretation of our result has to consider at least three consecutive sub-processes.. Dye Laser ~ 640 nm M Dichroic Mirror AOTF DM M Wedged Mirror Tube Lens Emission Filter CCD Camera FIG. 48. R. Chem. According to the rather limited photostability of GFPem we observe in most cases photo-bleaching after few exposures and in some cases also photoblinking. Schlesinger. 2002. 1758-1761 [3] M.3 minutes. in future we aim to incorporate fluorescent dyes into the elongating nascent chain in order to monitor co-translational folding events by the use of Förster resonance energy transfer (FRET) or by fluorescence anisotropy measurements. M. (ii) Folding rates of GFP are typically known from refolding studies. (i) First.. 83 . Zimmer. Here we demonstrated that co-translational folding can be monitored on single molecule level by employing surface tethered ribosomes. such like misfolding and aggregation. 32715-32718. T. and chromophore formation. The time course of emerging fluorescent GFPem molecules is satisfactorily fitted by a single exponential. Nierhaus. 102. As demonstrated here protein folding seems to be fast in our approach. Since the maturation time of the intrinsic GFP chromophore is in the order of minutes this approach does not allow to study faster events which are directly related to protein folding. whereas wild type GFP shows even longer maturation times in the order of 2 hours. [2] A. we identified several artificial peptide ligands from screening a phage displayed peptide library against lymphocyte specific kinase (Lck) SH3 domain [2]. 74-80). loops and alpha helices are colored red. and are mediators of protein-protein interactions [1]. SH3 domains recognize unique proline-rich peptides bearing the core sequence PxxP (where P = proline. Residues of the peptide with side chains are shown as stick model with electron density from the final 2Fo-Fc map.Crystal structure of PI3K SH3 domain in complex with an artificial peptide R. P=conserved residue proline. 28-33. J. Granzin1. Two 310 helices are shown within the loops in dark blue. The bound peptide has an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. In addition. D. yellow and blue. seen for the class I orientation (Figure1). The residues with their sidechains facing the protein are Arg4. p=proline preferred) (Figure 1). respectively. The role of SH3 domain is to mediate proteinprotein interaction via binding to proline-rich motifs in the target proteins enabling the formation of multimeric signaling complexes. The overall β-barrel fold of PI3K SH3 crystal structure is similar to that observed for the SH3 domains. Recently. we provide a comparative analysis of protein-ligand interactions that has helped us identify residues which play an important role in defining target specificity.to micromolar range. Batra-Safferling1. to shed light on the issue of specificity of the ligand binding to SH3 domains. 64-70. The studies are aimed to first. 1: Crystal structure of the PI3K SH3 domain in complex with ligand peptide PD1R. The peptides screened follow a Class I (+xxPxxP) consensus sequence with an average length of 12residues (+=basic residue. two short 310 helices and three loops (Figure 1). Our efforts remain to structurally investigate several different SH3 domains in the presence of artificial peptide ligands that bind in μM range. Considering the above mentioned crystal structures of PI3K SH3 and the published reports.7 Å reveals the type I ligand orientation. two unique regular helices (34-41. Ligand binding: The bound peptide adopts a lefthanded PPII helix conformation. Here we investigated the crystal structures of the SH3 domain from phosphatidyloinositol 3kinase (PI3K) in the presence of an artificial 12residue proline-rich peptide PD1R (HSKRPLPPL PSL) (Table1) [2]. PI3K SH3 domain is shown as ribbon structure where beta strands. x = any amino acid).7 Å resolution (Figure 1). Willbold2 1 2 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Institute of Structural Biology and Biophysics: Structural Biochemistry (ISB-3) Src homology 3 (SH3) domains are small protein modules (~60 residues). 54-60. we also attempted crystallization studies with another artificial peptide ligand PD1Y where the anchor residue at position P-3 is a tyrosine. we solved the crystal structure of SH3 domain from PI3K kinase in complex with an artificial peptide PD1R at 1. found in a number of intracellular signaling proteins. The protein structure consists of two orthogonal β-sheets containing five antiparallel strands (4-11. Pro7 and Pro10. Binding of these peptides is not specific and the binding constants vary in milli. FIG. The crystal structure reveals that the ligand is missing but the binding site is occupied by the residues Arg18 and Trp55 from the symmetry related molecule. The contour level is at 1σ. flanked by other residues specific to the domains. The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1. 50-53). Hoffmann2. S. In previous experiments. The amino acid sequence of the peptide PD1R is HSKRPLPPLPSL 84 . elucidate the molecular interactions involved in binding mechanism and second. The overall structure of SH3 domain shows minimal changes upon ligand binding. It is known from previously solved structures and from the sequence analysis that Trp55 is a highly conserved residue that contacts the ligand and is important in ligand binding. allowing the crystal packing where the ligand is omitted. [3] H. Gish. A.. Wiesehan. K. 365. Willbold. In a parallel attempt. D. [2]. However. C. Cell. and residues Pro7 and Pro10 correspond to the positions P0 and P3. we suggest that the binding mechanism involves two steps – first. Strangler. solution structure of hematopoietic cell kinase (Hck) SH3 domain in complex with PD1Y (Figure 2B) [3]. Cell boundary is shown in gray line. In future studies. [2] T. 71. Biochemistry. Arg4 lies sandwiched between the residues Arg18 and Trp55 of the protein. carrying a tyrosine at anchor position P-3 instead of an arginine (Table 1). Sequence and KD values of the artificial peptide ligands identified by Tran et al. Wiesehan. Trp55. Also shown is the side chain of residue Trp55 of SH3 domains. Hoffmann. 44.3. binding of the anchor residue and second. Stoldt. 359 (1992). critical for ligand binding. Schmidt. the ligand binding site is occupied by residues Arg18 and Trp55 of a symmetry related molecule where the side chains of arginine residues are sandwiched between the side chains of two tryptophan residues (Figure 2A). interacting with residues Tyr12 and Tyr73 of the SH3 domain. In the later structure. S. Pro70. Willbold. K. Hoffmann. In contrast. 15042 (2005). The stabilizing role of Trp-Arg-Arg π-π-stacking has been recognized in crystal structures for several receptors. structure based alignments show that residue Arg18 is not conserved and a great deal of structural variations are seen at this position upon ligand binding. readjusting the C-terminal residues that cause a kink in the peptide (Figure 2B). lysine at position P-3 substitutes the function of anchor residue. S. 2: A. T. the protein crystallized as free PI3K SH3 domain and not as PI3K SH3-PD1Y complex. B. Tran. Here. Pawson. As the residue arginine is absent at the anchor position in the PD1Y ligand. ligand is not present. we would investigate the role of non-conserved residues (like Arg18 of PI3K) which not only play a role in ligand binding. 85 . Jonas. T. It is plausible that the Trp-Arg-Arg stack formation (as seen in the complex structure) is entropically a favorable event. The residues reside on three well-define pockets: The Pro7 sits in the pocket formed from residues Tyr14. Aladag and D. we tried to co-crystallize PI3K SH3 domain with a modified peptide PD1Y. we show that upon substitution of residue arginine at the anchor position by tyrosine in the ligand leads to binding in solution but in the crystal structure. Asp21 and Asp68 where it forms salt bridges with Glu17 and Asp21 of the protein.1517 (2006). Luge. the protein prefers residue Arg18 of the symmetry related protein molecule instead.Sequence KD value (μM) 40 120 PD1R PD1Y HSKRPLPPLPSL HSKYPLPPLPSL TABLE 1. Based on our studies. We compared the PI3K SH3-PD1R complex crystal structure with our previously reported NMR FIG. binding of the polyprolines. E. but also in the selection of their respective ligands. M. Mol. J. Even though the KD values are in μM range. Interactions mediated by crystal lattice between the PI3K SH3 domain and the symmetry related molecule. The Trp-Arg-Arg interactions seen are similar to those observed between the anchor residue Arg4 of the peptide PD1R and residues Arg18 and Trp55 of SH3 domain in PI3K SH3-PD1R complex. Such non-conserved positions that participate in ligand binding are predicted to play a role in defining specificity for the ligand. and D. and Tyr73 of the SH3 domain. Tran. [1] T. where the residue Arg4 is at the anchor position P . and is surrounded by acidic residues Glu17. Super-imposition of PI3K SH3 bound PD1R (yellow) with HcK SH3 PD1Y(red). and G. Pro10 and side chain of residue Leu9 are both in a common pocket. Biol. 75 0. Rosenkranz1. and in the refolded state.1B).25 0. In principle.0 0. In vitro many of these proteins are well characterized by a reversible two state folding scheme. the majority of proteins in the cell belong to the class of larger multi-domain proteins which often unfold irreversible under in vitro conditions. as used for example in fluorescence correlation spectroscopy (FCS) studies. However.8 0. while at higher protein concentrations (~μM) refolding was hindered due to aggregation (Fig. In particular fluorescence based methods can contribute significantly to proceed in understanding fundamental principles of multidomain protein folding. Fitter1 1 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In addition. in the unfolded stated. working at low protein concentrations and employing single molecules studies are well suited to extend our knowledge on multi-domain protein folding. (B) Transitions as observed under unfolding and refolding conditions measured with CD spectroscopy at a protein concentration of about 3 μM.00 0 1 2 GndHCl [M] BLA_native BLA_unfol BLA_refol 3 BLA_unfolding BLA_refolding (C) norm G(τ) 0. As shown here. (C) Autocorrelation curves as measured with FCS for BLA in the native state.Studies on multi-domain protein folding T. Typically. 86 . orange. because of methodical reasons our existing knowledge about mechanisms and principles of protein folding results mainly from studies on smaller single-domain proteins. FCS is an easily applicable technique which needs only one fluorescent dye to be attached to the protein of interest. 1: (A) Schematic representation of the Bac. Extremely low protein concentrations (~ nM).00 fraction unfolded (B) 0. For protein folding studies on single molecule level mainly FCS and energy transfer techniques (FRET: Förster resonance energy transfer and PET: photoinduced energy transfer) have been employed. cyan) represents the domain structure.01 0. For α-amylase at least a partial refolding was observed only at extreme low proteins concentrations (~nM) as used in FCS. the application of single molecule techniques has an enormous potential for studying the intrinsically heterogeneous process of protein folding. can lower or even circumvent aggregation of unfolded states. The diffusion time of the fluorescently labeled protein through the confocal volume of a tightly focused laser beam allows to determine the hydrodynamic radius (Rh) of the protein. The color code (green. Therefore. this can make refolding studies feasible. Licheniformis α-amylase (BLA) structure. A promising approach in this respect is given by single molecule techniques which experienced tremendous methodological advances in recent years. J. In particular competing side reactions such as misfolding and aggregation of non-native states makes folding studies on larger multi. However. proper refolding is not taking place.2] (Fig. not only for multi-domain proteins. In prokaryotic and even more in eukaryotic cells the predominant fraction of the whole proteome belongs to the class of multi-domain proteins. the native state is compact and exhibits a smaller hydrodynamic radius as compared to the more expanded unfolded state.50 0.domain proteins often difficult or even impossible under in vitro conditions.1 1 time [ms] 10 100 FIG. which would be hampered by aggregation at higher protein concentrations [1. Here at low protein concentrations (~ 1nM) we observe at least a partial refolding (red line moves back to that for the native state). 1). (A) 1.4 0. This makes folding studies difficult or even impossible. A. I. in particular of the unfolded state. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies. J. ChemBioChem. Upon unfolding. (C) A typical time-course of the measured emission intensity as obtained from the integration of an individual spot. xx-xx [3] T. Moreover. is to encapsulate single molecules in surface-tethered nanocontainers (Fig. Rigler.. Life Sci. Fitter. Fitter. [1] K. Cell. Strucksberg. BBA. The fact that polymeric vesicles possess an extreme stability with respect to various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. T. 2009. (B) Wide field fluorescence image of surface tethered polymerosomes containing Atto655 labeled PGK.. The corresponding images (see B) were measured every 30 seconds with polymerosomes bound to cover-slides which were built-in a closed imaging chamber suitable for in-situ buffer exchange.g. 2007. 2A). In the case of PGK we were able to make use of PET due to the fact that Atto655 attached at a defined position within the protein structure is efficiently quenched by a tryptophan residue located in close proximity to the dye (low emission intensity). A major goal for future studies will be the focus on proteins that exhibit unfolding/folding transitions slow enough that trajectories of these transitions can be followed. a dye that exhibits pronounced photoinduced electron transfer (Fig. Fig. 2: (A) Scheme of an individual in a polymersome encapsulated protein labeled with a fluorescent dye. a structural expansion takes place and the average distance between the dye and the quencher is increased which results in a lower quenching efficiency (high emission intensity.Another approach to avoid protein aggregation. D. By this kind of encapsulation individual proteins are separated from each other and represent an ideal probe for single molecule studies. The permeability of triblock-copolymer membranes for GdnHCl and their resistance against structural disintegration at high denaturant concentrations ensure ideal conditions for their use in protein folding studies. Katranidis. M. T1/2. J. Fitter. 2A). The arrows indicate buffer exchange from native to unfolding conditions or vice versa. unlike in case of freely diffusing molecules 87 . 2C). Fig. polymerosomes prove to be permeable to GdnHCl and thereby ideally suited for unfolding and refolding studies with encapsulated proteins. Rosenkranz. Rosenkranz.10. We demonstrated here that proteins encapsulated in polymeric vesicles offer the possibility to study individual proteins for extended time periods. in solution. The encapsulation procedure provides a native-like environment for water soluble proteins without detectable hindrance of rotational reorientations. Atta. In order to immobilize individual protein molecules. 66(5). Gregor. At these conditions the folded and unfolded states have life times in the order of seconds and multiple successive unfolding/ refolding transitions can be observed with FRET for single encapsulated proteins. The high structural stability and considerable longevity of the polymerosomes qualifies them to be an ideal tool for single molecule studies. W. Enderlein. 1774(12). Meier and J. We demonstrate this with encapsulated phosphoglycerate kinase (PGK). 1591-1603 [2] J. Mol. P. Grzelakowski. which was fluorescently labeled with Atto655. At thermodynamic midtransition points (e. we encapsulate them in polymeric vesicles made of amphiphilic triblock-copolymers and tether the vesicles to a cover slide surface. pH1/2) in particular FRET based approaches provide structural details during folding and unfolding transitions of the proteins.H.702-709 Immobilizing biomolecules provides the advantage to observe them individually for extended time periods. 2009. GdnHCl1/2. 88 . Some of our target proteins are fibril forming or membrane proteins and are therefore not readily amenable to structural investigation. 89 . Furthermore. SARS and neurodegenerative disorders to study the molecular basis of these diseases and to investigate novel approaches to therapy. Our research purposes include e. the investigation of the structural basis of protein-protein and protein-ligand interactions to explore how viral proteins of HIV and SARS-CoV interact with host cell proteins to use and reprogram the host cell for viral replication and protection against the host immune system. X-ray crystallography are developed and applied.ISB-3: Structural Biochemistry Director: Prof. computational biology approaches in the fields of structural biology are performed. Dieter Willbold The research areas of the Structural Biochemistry group comprise the investigation of precise three-dimensional structures and dynamics of biologically and medically relevant macromolecules in order to fully understand their functions and their related cellular processes. e.g. To study these phenomena several molecular biology and biophysical techniques. fluorescence correlation spectroscopy as well as. liquid and solid state NMR.g. Research is focused on proteins that are involved in diseases like AIDS. ultra-sensitive assays potentially suitable for early and non-invasive diagnosis of neurodegenerative disorders like Alzheimer’s disease and prion diseases are developed. in cooperation with ISB-2. Moreover. 3 1 2 ISB-3: Structural Biochemistry Dept. Soluble Aβ polymerizes to form neurotoxic oligomers.D-peptides for diagnosis and therapy of Alzheimer’s disease S. We searched for peptides consisting solely of Denantiomeric amino acids (D-peptides) with strong binding to Aβ(1-42). β. Heinrich Heine-Universität Düsseldorf The “amyloid cascade hypothesis” assigns the amyloid-beta-peptide (Aβ) a central role in the pathogenesis of Alzheimer’s disease (AD). To improve diagnosis and treatment evaluation. consists mainly of extracellular Amyloid-β peptide (Aβ) deposits. AL 35294. In order to obtain the exact mirror image of a given protein (L-protein). A mirror image phage display approach was used in our lab to identify novel and highly specific ligands for aggregated Aβ(1-42). A. and deposits around neurons. which also fold to fibrils. The peptide was directly injected in the brain of the mice. More than 20 million people are affected worldwide.1: FITC-labelled D1 binds to Aβ plaques in the brains of transgenic APPswe-PSΔ9 mice in vivo. the D-enantiomeric form of the selected 12-mer peptide should interact with the Aβ peptide consisting of L-amino acids.and γ-secretase. Fig. These deposits spread in different areas of the brain and the "amyloid-cascade-hypothesis" assigns those Aβ plaques a central role in the pathogenesis of the disease. Wiesehan1. L. Mirror image phage display allows the use of phage display to ultimately identify peptides that bind specifically to a given target and consist solely of D-amino acids. Fibrillar deposits derived from other amyloidoses are not stained. it will mainly be developed as a molecular probe for in vivo imaging of amyloid plaque load in AD patients. Alzheimer´s disease (AD) is a progressive. the mice were sacrificed and the brains were investigated by histochemical analysis. So far. T. The histopathological hallmarks are protein deposits (senile plaques and neurofibrillary tangles) in the brain. 1. are developed. making use of Aβ-binding ligands visualizing amyloid plaques. Willbold1. neurodegenerative disorder which is characterized by loss of brain functions. Funke1. 90 . van Groen2. K. we used the D-enantiomer of Aβ as target and a 12-mer randomized peptide library displayed on the surface of M13 bacteriophages to be screened for D-Aβ-binding peptides. it is necessary to synthesize a protein with the same amino acid sequence but composed exclusively of D-amino acid residues (D-protein). Using mirror image phage display we identified two interesting D-peptides having different properties concerning Aβ cytotoxicity and Aβ aggregation. D1 binds to Aβ deposits in brains of AD transgenic mice (APPswe-PSΔ9) in vivo [2]. no method for pre-symptomatic diagnosis of AD is available. D-peptides are thought to be protease resistant and less immunogenic than the respective L-enantiomers. USA 3 Institute of Physical Biology. more resistant to degradation in animals and less or even not at all immunogenic as compared to L-amino-acid peptides. binds very specifically to Aβ aggregates with a binding constant in the submicromolar range. Kadish2. Here. In the study described herein. Aβ is a 4 kDa peptide of 39 to 43 amino acids derived from proteolytic cleavage of the amyloid precursor protein (APP) by two enzymes. D-Amino-acid peptides are known to be less protease sensitive. D1. The resulting peptide. Although D1 shows interesting properties in reducing Aβ cell toxicity and amyloid formation assays [3]. senile plaques. I. One week after injection. Such a D-protein can be used as a target for phage display selection like any other target. Cell Biology. For reasons of symmetry. a phage displayed peptide library with more than 1 x 109 different 12mer peptides was screened for peptides with binding affinity to the mirror image of aggregated Aβ(1-42) [1]. neuroimaging tools. Nagel-Steger3. University of Alabama at Birmingham. D. One of the typical hallmarks of AD. wheras peptide D3 has interesting therapeutical properties. As can be seen in Fig. Peptide D1 is being developed into a probe for in vivo imaging of amyloid plaques in the living brain. Therefore. [1] Wiesehan K. 4). These mice develop elevated levels of Aβ at four months of age and at the age of 5 months they show amyloid plaque pathology. processes. Unger E. Bucci E. inhibition of Aβ production and aggregation are often addressed for therapy development. During a second phage display selection with monomeric or low molecular weight oligomeric D-Aβ(1-42). Figure 4. USA) we carried out investigations on the effects of D3 on Aβ deposits in brains of transgenic mice (APPswe/PSΔ9). D3 is a good starting point for therapy development. 748-753 (2003). Kadish I. we obtained peptide D3. indicating that D3 changes the inflammatory properties of Aβ. Different concentrations of D3 were added to 10 µM Aβ samples or to samples without Aβ as controls. and coronal sections were cut through the brain. Percentages of cell viability were derived as follows: The 100 % value was obtained from cells treated neither with the respective peptide nor with Aβ. Please note the decrease in inflammation (activated astrocytes and microglia) surrounding Congo red-positive plaques in the D3 infused brain compared to the control brain.e. and coronal sections were cut through the brain. An inspection of the brain sections showed a significant reduction of the Aβ-load in the hippocampus and frontal cortex after treatment with D3 (see Fig. density of activated astrocytes and microglial cells) around remaining Aβ deposits revealed that the D3 treatment significantly reduced inflammation 91 . Animals were transcardially perfused at the end of the infusion period. Willbold D. 276-282 (2009). van Groen T. Wiesehan K. [4] Van Groen T.FITC-labelled In cooperation with Thomas van Groen (University of Alabama.2 % Triton-X. the value of 0 % was obtained by treatment of the PC12 cells with 0. Stöhr J.2: PC12 cell viability in absence or presence of Aβ and/or D3. Riesner D and Willbold D Prot. In recent studies. Sel. Saline (Control). Patt S. The Aβ/D3mixtures were incubated for 6 days at 37°C and 1:5 diluted into PC12 cell cultures. Therefore. [2] Van Groen T. 241-246 (2008). 3) [4]. Linke RP. Wiesehan K. [This article was evaluated by the Faculty of 1000 Biology as a "must read"] Detailed analysis of the inflammation (i. agents that interfere with early oligomerization processes might be valuable for therapy of AD. Nagel-Steger K. Funke SA. ChemMedChem 4. 120 ** ** cell viability (%) 100 ** * 80 60 40 20 0 0 10 25 no D3 10 µM D3 100 µM D3 1 mM D3 Aβ 1-42 concentration during aggregation (µM) Fig. D3 improved the cognitive performance of treated mice in the water maze assay after oral application (unpublished results). Fig. Birmingham. Cell viability was measured using MTT assay. which modulates Aβ aggregation and reduces Aβ cytotoxicity in vitro (Fig. prefibrillar aggregates and ADDLs) instead of fibrillar forms are the major toxic species in AD. We suggest an Aβ (1-42) modulating activity of the peptide as it clearly interacts with early intermediate assemblies of Aβ and promotes fibrillization (unpublished results). Nagel-Steger L and Willbold D ChemMedChem 3: 1848-1852 (2008). 3: Influence of D3 on Aβ load in brain tissue sections of transgenic APPswe-PSΔ9 mice. Animals were transcardially perfused at the end of the infusion period. Kadish I. Willbold D. Representative sections showing Congo red positive plaques the dorsal cortex are shown. Funke SA. The number of activated astrocytes and microglial cells (measured as density of GFAP and CD11b staining) near plaques of equal size was significantly less compared to the control brains (Fig. Buder K. [3] Wiesehan K. Des. Please note the decrease in Aβ staining in the D3 infused brain compared to the control brains. ChemBioChem 4.Although there is still controversial discussion if Aβ is the causative agent in AD. 21. one series of sections was stained with GFAP or CD11b. Recents studies indicate that soluble Aβ oligomers (also called protofibrills. Influences of D3 on inflammation in brain tissue sections of transgenic APP-PSΔ mice. D1 as a peptide control or D3 were infused into the brains of the mice for four weeks.. Stoldt M. Representative sections showing the hippocampus and dorsal cortex are shown. Schmitt B. 2). one series of sections was stained with W0-2 (anti human amyloid-β). Eng. Saline (Control) or D3 was infused in the brains of the mice for four weeks. purification. a short transmembrane region. C422S. C397S. We produced CD4tmcyt as part of a fusion protein containing an N-terminal decahistidine tag and the soluble protein ubiquitin (Fig. and a carboxy-terminal cytoplasmic tail (CYT). 1: Schematic representation of human CD4 (above) and of the recombinantly produced fusion protein (below). We obtained ~6 mg CD4tmcyt per liter of bacterial culture. Linker L2 contains PreScission and thrombin recognition sequences. purification.Human CD4: NMR structure and interactions of C-terminal domain M. CD4 features a large extracellular domain (EXT). The virus is internalized after binding of HIV-1 gp120 to the extracellular domain of CD4. including five cysteine residues.coli is very challenging. B. light orange: CYT). It often results in insoluble protein aggregates. S. 92 . isotope labelling. FIG.2. one transmembrane span (TM). Protease cleavage sites are indicated by asterisks. Willbold1. In addition to these functions. CD4 is the major receptor for HIV infection.6]. 1) [5. The CD4 T-lymphocyte coreceptor belongs to the IgG-superfamily and participates in T-cell activation and signal transduction. Wittlich1. D. Supplementing all buffers employed for cell lysis. and C430H at similar yield using the same strategy [6]. two additional viral proteins. 1). Recombinant production of transmembrane proteins in E. Specific binding of liposomereconstituted CD4tmcyt with the cytoplasmic domain of Vpu is demonstrated. are necessary and sufficient for down-regulation of CD4 by Nef [2-4]. and a cytoplasmic domain of 40 amino acids (Fig. and enzymatic cleavage of the fusion protein with a dedicated mixture of non-ionic surfactants was crucial for increasing solubility of the protein and for the exceptionally high yield of purified CD4tmcyt. C420S. CD4tmcyt contains the 60 C-terminal amino acid sequence of wild type human CD4. Human CD4 consists of an extracellular region of 371 amino acids. We plan to introduce single cysteines into this mutant for attachment of ESR or fluorescence labels. Heinrich-Heine-Universität Düsseldorf The type 1 transmembrane glycoprotein CD4 of 58 kDa plays a key role in the adaptive immune response. Following infection. containing the transmembrane and cytoplasmic domains of CD4. a decahistdine tag. Physical interactions of HIV-1 accessory proteins Nef and Vpu with the cytoplasmic tail of CD4 result in downregulation of CD4 from the surface of infected cells. In contrast. and numerous obstacles during protein refolding and membrane reconstitution. The NMR structure of CD4tmcyt in membrane mimicking micelles is presented. with special emphasis on the dileucine motif at positions 413 and 414. and the 11 residue linker L1 (SSGHIDDDDKH). but bind to the cytoplasmic domain of CD4. interact with CD4. The authentic CD4 residues 372 to 433 are highlighted (dark orange: TM. CD4’s ectodomain serves as main receptor of human immunodeficiency virus (HIV) and binds the HIV-1 envelope glycoprotein gp120. Residues 407 to 418 in the cytoplasmic tail of CD4.2. Vpu and Nef.2 1 2 ISB-3: Structural Biochemistry Institute of Physical Biology. W. We also produced the cysteinefree protein CD4mut with substitutions C394S. The CD4 sequence relevant for this activity is located between amino acids 402-420 [1]. and membrane reconstitution of CD4tmcyt. The N-terminal tag of the fusion protein consists of yeast ubiquitin preceded by a dipeptide (MG). respectively. low protein yield. Here we present an efficient protocol for recombinant production.2. The amino acid sequence of the 70 residue CD4tmcyt protein obtained after PreScission cleavage is shown in the lower part. Our goal is the detailed characterization of the structure of the membrane-anchored Cterminal domain of CD4 in complex with Vpu and Nef. Koenig1. Nef acts at the cell surface to mediate the internalization and lysosomal degradation of CD4. Hoffmann1. Vpu-induced degradation of CD4 molecules in the endoplasmatic reticulum requires both proteins to be inserted into the same membrane. 3: Schematic representation of the transmembrane and cytoplasmic helices of CD4mut in a detergent micelle. 67. J. Lenburg. In contrast. and D. Biophys Acta 1768. L. respectively. E. J. J. Wiesehan. The composition of supernatant (left column) and pellet fractions (right column) was analyzed by analytical RPC using a gradient of buffers A and B (dashed line).S.5 mg – middle. Chen. F. 55. Karczewski.Both CD4tmcyt and CD4mut were successfully reconstituted into liposomes. W. B. Strebel. Willbold. was quantified by reversed phase HPLC (Fig. CD4mut (2). K. [1] M. and K. transmembrane helix. R. 198 (2007) [6] M. R. 2). The location of CD4 residues relative to the micelle was studied with paramagnetic probes: Supplementing the buffer with manganese ions (MnCl2) strongly broadens resonances in the cytoplasmic helix and in the interhelical loop. Willbold.Virol. Cell 76. They extend from M372 to V395 and from M407 to R412. Position of the VpUcyt (1). Garcia. Baur. The amount of soluble Vpucyt in the supernatant and in the membrane fraction. Konner. Our data indicate specific binding of the recombinant CD4 domain to Vpucyt. 75. The ribbon diagram reflects the length of the two helices 1 1 derived from H – H distances and secondary shift analysis. FIG. L. Wittlich. 2949 (2007) 93 . Willbold. A. FIG. Virol. respectively. 1 mL each) containing varying amounts of reconstituted CD4mut (0 mg – above. and D. Briese. 3877 (1993) [2] C. N. incorporation of 16-doxylstearic acid into the hydrophobic core of the micelle affects only resonances in the transmembrane helix. M. 2: Analysis of CD4mut Vpucyt interaction in POPC membranes using centrifugation followed by reversed phase chromatography. S. J. and POPC (3) peaks are indicated. 2 mg – below) were incubated with 50 μM Vpucyt. in good agreement with the predicted transmembrane and cytoplasmic helices. 3960 (2001) [5] M. Trono. Hoffmann. Koenig. Two α-helices were identified based on NOE1 1 derived H. Willey. K. The position of the two helices with respect to the micelle was deduced from signal broadening caused by paramagnetic probes. W. 68. but shows little effect on resonances of the [3] S. Protein Expr. Koenig.H distances and secondary chemical shifts. Landau.Purif. Maldarelli. Binding of Vpucyt (the C-terminal amino acid residues 39-81 of Vpu) to the CD4 fragments was demonstrated with a liposome centrifugation assay [5. 3092 (1994) [4] A. Wittlich. 835 (1994) We determined the conformation of CD4mut in membrane mimicking dodecylphosphocholine (DPC) micelles using high resolution NMR (Fig. Dispersions of POPC liposomes (10 mg POPC per sample. a novel phospholipid-based model membrane. and J. N. Y. Our next goal is incorporation of membrane proteins like CD4tmcyt into nanodiscs. M. M. and D. B. and D. 3) [6]. The two cylinders surrounding the helices symbolize the space requirement of the amino acid side chains. 0.6]. Landau. Lenburg. The mutual orientation of the two helices remains undetermined due to lack of long range NOEs. V. followed by NMR structural studies and interaction analysis. J. Fully hydrated liposomes were separated from supernatant by centrifugation. Anderson. Virol. R. Aiken. Preusser. Biochim. T. Riesner2 1 2 ISB-3: Structural Biochemistry Institute of Physical Biology. soluble but β-sheet rich oligomers of a minimal size of 12–14 PrP molecules. Kaimann2. We assume that the α-helical part is identical or close to that which is also present in the fibrillar structure. the lower part with added NaCl. The structure of the intermediate state PrP* is decisive to switch aggregate formation from the polymorphic FIG. Addition of 250 mM NaCl shifts PrP into a still soluble state that allows fibril assembly to proceed over several weeks.Mechanism of prion protein conversion and assembly E. Heinrich-Heine-University Düsseldorf Prion diseases are a unique group of neurodegenerative diseases because they are know as transmissible. G. Therefore. well characterized intermediates and precursor states during the conversion process as well as kinetic studies of spontaneous and seeded fibrillogenesis are described. we studied the structure of dimers in more detail. The upper part of the figure represents the conversion without added NaCl. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and molecular mass as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation. respectively [4]. To obtain information on the conformation of the dimer. (Figure according. 1 the different structures are represented schematically together with their CD-spectra and the EM picture of the polymorphic aggregates. With these methods α-helical monomers. In 0. Studies using analytical ultracentrifugation demonstrated the existence of a monomer-dimer equilibrium.[3]) The studies described above indicated a critical role of PrP dimers in the conversion process consistent with research results from other groups. Exemplarily for the methods used to analyse these structures the CD-spectra are presented beneath the different intermediates. The molecule populations analysed within the in vitro conversion system are summarized. respectively. Furthermore.2. In Fig. Panza. we used the covalent crosslinker EDC (1-ethyl-3-(3dimethylamino-propyl) carbodiimide). Birkmann1. we called this state fibril precursor state and characterized it by different methods. Different conformations could be established.2% SDS PrP is present in an α-helical and partially random coil conformation denoted with α/R. 1: Molecule populations involved in the in vitro conversion of recPrP(90-231) induced by lowering SDS concentration. They can occur both spontaneously and genetically caused. Our in vitro conversion system is based on the solubilization of PrP in low concentrations (0. soluble α-helical dimers.2% w/v) of sodium dodecyl sulfate (SDS) under otherwise physiological conditions. recPrP(90-231) in 0.2. and insoluble amorphous aggregates of β-sheet rich structure were observed.06% SDS 10 mM phosphoric buffer pH 6. The mechanism of spontaneous prion protein conversion was studied systematically with recombinant (rec) PrP. J. A high activation barrier was found between the α-helical dimers and the β-sheet rich oligomers. which forms 94 . We selected conditions which stabilize dimers or drive the dimer-oligomer equilibrium completely to the side of dimers. The results determined with our in vitro conversion system and the derived mechanistic models are presented as a brief summary. D. D. Most extended and systematic studies were carried out with hamster recPrP at neutral pH.8 without additional NaCl is 100% dimeric. The conversion is induced by diluting the SDS. Consequently. We investigated the conversion process by in vitro studies using recombinant PrP and natural PrPSc. Stöhr2. Next to the aggregated states the electron micrograph of these structures are shown. CD spectroscopy showed a mixture of α-helical and random coil secondary structure. Willbold1. pathway into the fibrillar pathway. The infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrPC into Sc the pathological and infectious isoform PrP [1]. 6 x 10 brain equivalents per μl (x). The fibrillization conditions were identical to those used for spontaneous fibrillization. 3: Seeded fibril formation Sc RecPrP (80 ng/μl) seeded with purified PrP (diamonds) forms amyloid fibrils readily compared with controls: -4 recPrP + uninfected 1. [4] Kaimann T. glycosyl groups. [1] Prusiner SB. When the cross-linked sites were used as structural constraints. 2: Model of the prion protein dimer. Eigen M & Riesner D (2008) Proc. N-termini. Prusiner SB. FIG. Birkmann. FIG. (2008) Prion. but the principle conversion mechanism could be approved as described above with recPrP. Sci. Brandt B. the structure of segment 125–228 (cyan) is taken from the NMR analysis reported in the literature. NagelSteger L. Wille H. (2008) Biochem. Sci. USA. 376. 2 (2). Riesner D. 13363-13383. The structural model (Fig. Natl. purified PrP alone (dashes). Stöhr J. molecular modelling calculations yielded a structural model for PrP dimer and its monomeric subunit including the folding of amino acids 90–124 in addition to the known structure of the core domain (Fig. Commun. E.and intramolecular bonds between directly neighboured amino and carboxyl groups. thick blue arrows. During the enhanced fibrillization a lag phase is followed by exponential growth and saturation. 2) is in agreement with GPI-anchoring of the dimer to the membrane. Höltje H-D and Riesner D. Metzger S. Papathanassiou D. Fibrillization of PrP without seeds takes up to several weeks. 105 (7). Weinmann N. (Figure rearranged according [2]) To compare the in vitro conversion and the seeding efficiency in respect of differences in different species. [3] Birkmann E & Riesner D. Mol. Kaimann K.inter. and thin blue arrows. (2007) J. Panza G. Natl. 95 . whereas the glycosyl groups and the N-termini are directed into the water phase. Biol. Acad. Dumpitak C. 493-497 As a model for the infection process fibril formation Sc was also studied when induced by seeds of PrP . 2). 95. 582-596 [5] Panza G. USA. whereas seed-enhanced fibrillization was achieved within hours to days (Fig. The bonds were identified by tryptic digestion and subsequent mass spectrometric analysis. Intraand intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified showing that the N-terminal amino acids (90–124) are not as flexible as known from NMR analysis. recPrP + PrP after seeding assay. we adopted the in vitro conversion system to bovine recPrP [5]. Sc We used natural PrP purified from brain homogenate as seeds. Red arrows represent the glycolipid anchor. (1998) Proc. Sc recPrP alone (triangles). Demonstrated by arrows the two glycolipid anchors are directed to the lower side where the membrane would be present. RecPrP was applied as substrate. Acad. Biophys. Birkmann E. We monitored and quantitatively described the kinetics of seeded fibril formation. Birkmann E. Res. (Figure according [4]) [2] Stöhr J. Molecular dynamics simulation with the dimeric structure after release of the crosslink constraints indicated the domain formed by amino acids 90–124 of both momomers to be intrinsically stable. We could observe some differences in the structure of the fibril precursor state. 67-72. The structure of segment 92–124 (blue) is the result of [4]. Willbold D. Weiss J. 3). Electron micrographs: PrP Sc aggregates after NaPTA precipitation. 373 (4). Fibril formation was monitored by using the ThT Sc fluorescence assay. Stereo presentation of the model for the PrP dimer. 2409-2414. Kuhlmann K. the overall morphology of amyloid fibrils in general depends strongly on the fibrillization conditions. and significant acceleration of fibrillization has been observed for the mutants A53T and E46K. M. which is accompanied by the formation of cytosolic filamentous inclusions called Lewy bodies. all three mutants are natively unfolded. Stefan Becker3.Solid-state NMR reveals structural differences between fibrils of wild-type and mutant α-synuclein Henrike Heise1. FIG. a 140residue protein. In the following. the most common neurodegenerative movement disorder. Three point-mutations of α-synuclein have been related to familial earlyonset Parkinson’s disease. whereas the main core region is highly rigid and rich in β-sheets. have been obtained and characterized [1]. Soledad Celej3. We have investigated the structure and dynamics of fibrils from full-length α-synuclein and of its disease-related mutant A53T by highresolution solid-state NMR spectroscopy. including pH. Parkinson’s disease. 2. Upon fibrillization of wild-type α-synuclein at physiological temperature and pH. and E46K as causes of autosomal dominant Parkinson’s disease. However. Avishay Pelah3. as differences in aggregation behavior and morphology with respect to the wild-type form may yield valuable insights into the fundamental processes underlying amyloidogenic diseases. at least two different polymorphic forms. one consisting of straight and the other of irregularly twisted fibrils. Ashutosh Kumar3. 1: 2D C/ α-synuclein fibrils temperatures of ~ Homonuclear mixing diffusion for 20 ms. 13 13 C correlation spectra of A53T recorded at effective sample 0 °C (red) and -20 °C (black). Jovin3. Thomas M. salt concentration and agitation of the solution. which in its native conformation is largely unfolded. The Netherlands The 140 residue protein α-synuclein is able to form amyloid fibrils and as such. and the supramolecular arrangement was found to be a superpleated β-sheet with parallel and in register alignment of β-strands [2] (Figure 2b). Since their discovery. In both cases the C-terminus was found to be flexible and unfolded. was achieved by proton-driven spin 96 . Göttingen 4 Utrecht University. Compared to fibrils from wild-type α-synuclein. temperature. whereas the C-terminus was flexible and unfolded in both cases. Dietmar Riedel3. As in the case of wild-type α-synuclein. Heinrich-Heine-Universität Düsseldorf 3 Max-Planck-Institute of Biophysical Chemistry. The central role of α-synuclein aggregation in the etiology of Parkinson’s disease has been further corroborated by the identification of a locus triplication of the gene encoding α-synuclein as well as the three point-mutations A53T. Electron Microscopy and Atomic Force Microscopy. The major constituent of these inclusions consists of fibrillar α-synuclein. A30P. we report studies on fibrils grown from the diseaserelated A53T mutant of α-synuclein by solid-state NMR Spectroscopy [3]. These results demonstrate that a disease-related mutant of α-synuclein differs in both aggregation kinetics and fibril structure. is caused by the loss of dopaminergic neurons in the substantia nigra. is the main component of protein inclusions involved in Parkinson’s disease. the well-ordered β-sheet region is extended. and Marc Baldus4 1 2 ISB-3: Structural Biochemistry Institute of Physical Biology. Studies of wild-type α-synuclein by solid-state MAS NMR-spectroscopy have revealed a β-sheet rich core region spanning residues from at least 38 to 94 for the two different polymorphic isoforms. the three disease-related mutants have been the subject of intense study. Becker. H. F. J. and a highly flexible and unfolded C-terminus.. USA. H. Herzig. Intrinsic dynamics of the protein were probed by spectroscopy at different temperatures as well as by a combined mobility filter experiment specific for mobile regions of immobilized molecules. Young.P. the low-temperature spectrum displays some additional sets of signals. Fichtner. submitted (2009). F. Heise. Voigt. [1] H. Becker. 12965. Biomol. S.H. indicating that the fibrils contain regions with increased mobility and structural elements other than β-sheet. For comparison. Only torsion angle pairs where at least 8 hits were found are plotted. 380. whereas the N-terminus lacks high mobility as of residue 22. Acad. in fibrils of both the mutant and wild-type α-synuclein. especially in the region typical for non β-strand secondary structures: In hydrated amyloid fibrils. Eimer. Mol. β-strands determined earlier for both wild-type forms23 are given together with the β-strands of A53T. Heise.. Karpinar. A. which is neither part of the flexible C-terminus nor of the rigid core region. S. [5] Cornilescu. S. recorded above and below the freezing point. almost all resonances indicative of a β-sheet conformation are in rigid protein compartments. G. Fibrils consist of a well-ordered rigid core region rich in β-sheets. Jäckle. Heise. in a recent study. 179-189 (2008). Heise. J. different regions of a protein may exhibit diverse residual mobility. 9. H. A.M. G. 15871-15876 (2005). W. Griesinger.G. [4] O. In contrast. whereas the N-terminus extending from residue 22 is rigid. S. D. M. Soc. Balija. the C-terminus is unfolded and highly dynamic. Freezing the sample reduces the overall mobility such that flexible parts may give rise to inhomogeneously broadened signals.In Figure 1. Zweckstetter. NMR 13. Heise. but belongs to a region of intermediate mobility. and solid-state NMR spectra based on dipolar transfer mechanisms often show only a subset of all resonances in the protein. Alternatively. Am. Andronesi. ChemBioChem. and M. Andronesi.B. Riedel. Kügler. M. Pelah. Braus. two 13C/13C correlation spectra of A53T fibrils. G. A. 127. depending on the degree of disorder. Biol. Opazo. where proline residues were inserted into β-strands in order to retard fibrillization and shift the equilibrium towards early monomers. The striking difference of the A53T compared with wildtype α-synuclein is an increase in the β-strand character of two leucine residues at the edges of the core region. Sci. and Bax. Hoyer. S. D.S. Kumar. B. well-ordered β-sheet rich core region towards at least residue L38 and L100. Schulz. [3] H. [6] D. Baldus.C. together with the error bars. M. Upon freezing the sample. Similar to our previous results obtained on wild-type α-synuclein. Riedel. D. 289-302 (1999). FIG. while the intensities of resonances in β-strands do not increase unexpectedly. [2] H.B. Becker. the C-terminus extending from at least residue 107 is flexible. Taschenberger. M.. b) superpleated β-sheet with in-register parallel alignment of β-strands The results of our investigations are summarized in Figure 2 a. Chem. Baldus.C. 444-450 (2008). For example. The accelerated aggregation kinetics itself could result in a different pattern of molecular assembly characterized by an extended β-sheet core region. A. K. Jovin. Proc. O. Baldus. S. However. These results provide important insights into the effect of changed aggregation properties on the final fibril morphology of one disease-related mutant of α-synuclein. are displayed. Falkenburger. While the crosscorrelation signals in the high-temperature spectrum are narrower and better resolved. Becker. C. T. Seidel. obtained on two design mutants A56P and A30P/A56P/A76P. Riedel. S. Delaglio.H. flexible regions can selectively be detected by applying adequate mobility filters in the MAS NMR experiments [4]. 102. Celej. Baldus. J. Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase toxicity in Parkinson`s disease models. and M. Further studies of this nature may help to delineate the relationship between the etiology of Parkinson’s disease and protein misfolding.2974 (2005). indicating an extension of the rigid. A. Kumar. Natl. 2: a) Backbone angles obtained with TALOS [5] analysis of the chemical shifts. H. the low-temperature spectrum indicates the existence of at least one valine residue with a chemical shift typical for α-helical conformation. J. our results. L. the N-terminus was found to be flexible as well [6]. and M. A. According to 97 . resonances with random coil and α-helical secondary chemical shifts appear in the spectrum. Site-specific resonance assignments for a large part of the amino acids in the core regions were obtained from 2D homonuclear 13C/13C and heteronuclear 15N/13C correlation spectra. and Parkinson’s disease are neurodegenerative diseases that are characterized by the formation of protein aggregates during progress of the disease. is yet available. Only aggregates and oligomers but not monomeric proteins are detected. 2. B) Scanning scheme. 2. It is still not known whether these aggregates are causative for or symptoms of these diseases. PrPC undergoes a conformational change during a posttranslational process. leading to altered physicochemical properties such as aggregation. It is quantifying the number and size of aggregates simultaneously labelled by two different antibodies for dual colour FIDA [1]. Following infection. bovine spongiform encephalopathy (BSE) in cattle and Scrapie in sheep. To understand disease-associated or causative mechanisms in respect to protein aggregation. To increase the sensitivity. 3] (Fig. whereas the risk of AD increases dramatically in individuals beyond the age of 70. Neither a causal therapy for recovery nor a reliable early diagnosis. A. confusion and a variety of cognitive disabilities. Riesner2. Wang1.Surface-FIDA: Single particle detection as diagnostic tool E. Henke1. L. The cellular. non-pathological isoform of PrP (PrPC) is present in most tissues and is most abundant in the central nervous system. D. The latest development of this test system is based on fluorescence microscopy using fluorescence intensity distribution analysis (FIDA). Today. Therefore. particles were concentrated in the two dimensional space by immobilizing it to capture antibodies on the surface of the slide. S. i. Prions are the causing agent of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt–Jakob disease (CJD) in man. These diseases are characterised by an abnormally folded form of the host-encoded prion protein (PrP). Alzheimer’s disease. insolubility and β-sheet rich secondary structure. The infectious agents of prion diseases are composed 98 . 1). 1: Surface-FIDA A) Scheme of surface-FIDA. 1 2 ISB-3: Structural Biochemistry Institute of Physical Biology. 3]. Birkmann1. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which is characterized by memory loss. AD is the most prevalent dementia affecting nearly 2 % of the population in the western world. In this project we introduce a new diagnostic tool to count and specify even single protein aggregates. because the according diseases are of particular interest as prion FIG. Willbold1. Sc This pathological isoform is designated PrP . called surface-FIDA [1. The major component of the amyloid plaques is the Amyloid-β (1-42) peptide (Aβ). Funke1. O. we established a highly sensitive and specific tool to detect even single protein aggregates. Prion diseases. which could improve present therapeutically or preventional approaches. Laser beams are scanning the surface systematically so that even single particles are detected [2. Within this project we focussed on the detection of PrP and Aβ particles. In both diseases protein aggregates occur during disease progression. D. C) Principal of particle counting: Fluorescence peak caused by the labelled aggregate. Heinrich-Heine-University Düsseldorf diseases are transmissible and AD is the most prevalent dementia. an ultrasensitive tool to quantify these diseaserelated aggregates is required. Many studies show that aggregates or even oligomers of the according proteins are neurotoxic and thus may lead to neurodegeneration.2. F. We successfully established surface-FIDA as a diagnostic tool for prion diseases [2].e. the most reliable diagnosis of AD is the post mortem identification of amyloid plaques and neurofibrillary tangles in the respective brain. Bannach2. primarily of the pathogenic isoform of the prion Sc PrP . In contrast to its cellular isoform, the pathogenic isoform PrPSc forms insoluble aggregates. Hitherto accredited prion tests use the proteinase K (PK)-resistance of PrPSc as a marker for the disease. These prion tests offer only a limited sensitivity because of varying portions of disease related aggregated PrP which is not PKresistant. Therefore prion protein aggregate detection which does not rely on PK-digestion is favourable because it allows detection of both, PKSc resistant and PK-sensitive PrP , aggregates. We could successfully verify our novel test system for correct diagnosis of Scrapie infected hamsters as well as BSE-infected cattle in the clinical stages of diseases. Furthermore, we were able to detect PrP aggregates in the cerebrospinal fluid (CSF) of BSE-infected cattle for the first time [1] (Fig. 2). The use of three different antibodies (capture and two detection probes) but with overlapping epitopes delivered the best specificity within the assay [4]. A B FIG. 2: Detection of PrP aggregates in cerebrospinal fluid (CSF) of BSE-infected cattle by surface-FIDA Surface-FIDA of BSE-infected and healthy cattle CSF. In the histograms the mean values of burst number detected within the surface-FIDA measurements are shown. Different capture probe combinations of the antibodies D18, Saf32 and 12F10 are used. CSF samples from two different BSE-infected cattle (dark grey) and two different healthy controls (light grey) were analysed. The number of peaks varied independently of the labelling grade of the probes FIG. 3: Detection of Aβ aggregates in CSF of AD patients A) The sensitivity of the assay was determined by dilution of the aggregates in CSF. For all measurements (three measurements ± standard deviation), burst number detected by 2D-FIDA during 0.5 min are shown. Fluorescence labelled antibodies 6E10 and 19H11 were used as detection probes. B) 2D-surface-FIDA was applied to 20 µl crude CSF of AD patients and control patients not affected by AD. Number of bursts (three measurements ± standard deviation) obtained by 2D-FIDA during 0.5 min are shown. [1] Birkmann E, Henke F, Weinmann N, Dumpitak C, Funke SA, Willbold D, Riesner D. (2007) Counting of single prion particles bound to a capture-antibody surface (surface-FIDA). Vet. Microbiol. 123, 294304. [2] Birkmann E, Schäfer O, Weinmann N, Dumpitak C, Beekes M, Jackman R, Thorne L, Riesner D. (2006) Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion. Biol. Chem. 387, 95102. [3] Funke SA, Birkmann E, Henke F, Görtz P, LangeAsschenfeldt C, Riesner D, Willbold D. (2007) Single particle detection of Abeta aggregates associated with Alzheimer's disease. Biochem. Biophys. Res. Commun., 364 (4), 902-907 [4] Birkmann E, Henke F, Funke SA, Bannach O, Riesner D, Willbold D. (2008) A highly sensitive diagnostic assay for aggregate-related diseases e.g. prion diseases and Alzheimer’s disease. Rejuvenation Res. 11(2), 359-363 [5] Funke SA, Birkmann E, Henke F, Görtz P, LangeAsschenfeldt C, Riesner D & Willbold D (2008) An ultra-sensitive assay for diagnosis of Alzheimer’s disease. Rejuvenation Res. 11(2), 315-318 We also established the surface-FIDA assay for the detection of Alzheimer’s Aβ aggregates. We showed that the assay is sensitive enough to detect aggregates in the picomolar range. First measurements show a clear distinction between AD diseased people and non-demented controls by analysing CSF [3, 5] (Fig 3). During the next steps we will adapt the highly sensitive test system for diagnosis of human prion diseases like Creutzfeldt-Jakob disease and other aggregate related diseases, e.g. Parkinson’s disease. Furthermore we will develop surfaceFIDA to an imaging assay. 99 NMR structure of the M.Loti K1 channel cyclic nucleotide binding domain S. Schünke 1 2 3 4 1,3 , M. Stoldt 1,3 , K. Novak , U. B. Kaupp 2 2,4 , D. Willbold 1,3 ISB-3: Structural Biochemistry ISB-1: Cellular Biophysics Institute of Physical Biology, Heinrich-Heine-University, Düsseldorf Center for Advanced European Studies and Research (Caesar), Bonn Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, play crucial roles in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels, is not well understood. We report the solution structure of the isolated CNBD of a cyclic nucleotide-sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide antiparallel β -roll topped by a helical bundle comprising five α-helices and a short 310 -helix. In contrast to the dimeric arrangement (“dimerof-dimers”) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other. Ion channels activated by cyclic nucleotides play key roles in neuronal excitability and signaling of visual and olfactory neurons. They belong to two subfamilies: Cyclic nucleotide-gated (CNG) channels, and hyperpolarization-activated and cyclic nucleotidegated (HCN) channels. Both channel types share a carboxy-terminal cyclic nucleotide-binding domain (CNBD). HCN channels are activated by hyperpolarization and their activity is modulated by cyclic nucleotides. In contrast, CNG channels are voltage independent and require cyclic nucleotides to open. Binding of cyclic nucleotides promotes the opening of the channel. Probably, a conformational change in the CNBD is propagated to the pore. Recently, a prokaryotic cyclic nucleotide-sensitive K+ -channel, designated MloK1, has been identified in Mesorhizobium loti. MloK1 harbors six transmembrane domains (S1-S6), a “GYG” signature sequence for K+ selectivity, and a conserved CNBD is connected via a short C-linker to S6 (Fig. 1). The longer C-linker (∼80 residues) of mammalian CNG channels is important for relaying the binding signal to the channel gate. Crystal structures of mammalian HCN channel CNBDs revealed that neighbouring C-linkers contribute virtually all contacts between subunits in the tetrameric protein. The crystal structure of the isolated CNBD of MloK1 suggested that subunits are organized as dimers. The dimer interface formed by the short linker has been proposed to be involved in channel gating [1]. Subunit topology and assembly of the full-length MloK1 cyclic nucleotide-gated K+ channel. MloK1 consists of four subunits. Each subunit encompasses six transmembrane segments S1-S6 (yellow) and an intracellular CNBD (shown in ribbon representation). FIG. 1: However, an electron microscopy study of the complete channel reveals a four-fold symmetry of subunit arrangement [2]. The CNBDs appear as independent domains separated by discrete gaps, suggesting that CNBDs are not interacting with each other. Furthermore, the MloK1 channel and the isolated CNBD bind cAMP with similar affinity in a non-cooperative fashion [3]. Here we study the solution structure of the monomeric CNBD in complex with cAMP by nuclear magnetic resonance (NMR) spectroscopy (Fig. 2) and compare it with the structure in the dimer (Fig. 3). 100 be set apart from its vertebrate cousins by the lack of a full-blown C-linker that coordinates intersubunit contacts. This conclusion is supported by an EM study of the complete MloK1 channel. An important feature of the EM structure is that the four CNBDs are separated by discrete gaps and that four isolated CNBDs could be modelled into the electron density map. This structure predicts that binding sites act independently. The C-linker contact observed in the crystal structure was possibly enforced by the packing of dimers in the crystal and that these contacts seem to be functionally irrelevant. In fact, cAMP and several analogues bind noncooperatively to the monomeric CNBD and the tetrameric full-length MloK1 with virtually identical high affinity [3]. Together with the presented solution structure, this shows that MloK1 cyclic nucleotide binding sites are functionally independent of each other [3, 5]. Solution structure of the isolated cyclic nucleotidebinding domain. A: Superposition of the backbone traces and all cAMP atoms of the family of 15 NMR structures with the lowest CYANA target function. Backbone atoms of the N- and C-terminal ends (residues Q216 to V218 and A351 to A355) are not shown and were not used for least-square superposition of the structures. B: Ribbon representation of the CNBD structure with the lowest target function. The cAMP ligand is shown as a stick model. Secondary structure elements are labelled. FIG. 2: The solution structure is very similar to the structure of a monomer in the dimer crystal [1]. The comparison of all CNBD backbone coordinates (residues V218 to G350) between solution and crystal structure results in a r.m.s. displacement value of 0.21 nm. However, the coordinates for the N-terminal residues (V218 to P241) which represent the α1-helix, 310 helix and associated loop regions differ remarkably. The α1-helix region (G221 to A231) in the solution structure is a straight helix without bending (Fig. 3). In the crystal structure, however, residues R220 to N226 of α1 are bent and form the dimer interface. The two most important insights of our study [4, 5] are that (1) the CNBD even at the high concentration required for NMR measurements is a monomer and that (2) the solution structure, except for the Nterminal C-linker region, is similar to the monomer structure in the dimer crystal structure. The much longer C-linker region of vertebrate CNG and HCN channels is involved in intra- and intersubunit contacts and contributes virtually all contacts between the subunits in the tetrameric crystal structures of CNBDs from two different HCN channels. The formation of dimers and tetramers from monomeric CNBDs requires cAMP, suggesting that rearrangement of the C-linker interface represents an important gating event. Thus, the MloK1 channel seems to Comparison of NMR and crystal structure. A: Comparison of the solution structure (blue) and the crystal structure of the CNBD (light gray). Part of the second domain in the dimer crystal structure that is involved in the dimer interface is depicted in green. Alignment of the solution and crystal structure backbones (residues V218 to G350) yielded an r.m.s. displacement of 0.21 nm. A comparison of the solution and crystal structure excluding N-terminal residues (V218 to P241) results in an r.m.s. displacement value of 0.12 nm. B: CNBD backbone comparison of the monomeric solution structure (shown in blue) and the backbone of the crystal structure dimer (shown in red). FIG. 3: [1] G. M. Clayton, W. R. Silverman, L. Heginbotham and J. H. Morais-Cabral, Cell 118, 615-627 (2004). [2] P. L. Chiu, M. D. Pagel, J. Evans, H. T. Chou, X. Zeng, B. Gipson, H. Stahlberg, and C. M. Nimigean, Structure 15, 1053-1064 (2007). [3] A. Cukkemane, B. Gruter, K. Novak, T. Gensch, W. Bonigk, T. Gerharz, U. B. Kaupp, and R. Seifert, EMBO Rep. 8, 749-755 (2007). [4] S. Schünke, M. Stoldt, K. Novak, U. B. Kaupp, and D. Willbold, Biomol. NMR Assign. 1, 179-181 (2007). [5] S. Schünke, M. Stoldt, K. Novak, U. B. Kaupp, and D. Willbold, EMBO Rep., Manuscript Accepted (2009). 101 The interaction of this high-affinity ligand with GABARAP was studied using NMR spectroscopy and X-ray crystallography [2]. hydrophobic side chains involved in complex formation are indicated. Combining pulldown experiments and data mining. deed. which play an essential role in complex formation with native ligands. free indole and several derivatives were chosen as probes to characterize the binding properties of GABARAP by nuclear magnetic resonance (NMR) spectroscopy [1]. While the three-dimensional structure of GABARAP itself has been determined. Mohrlüder1 . Proteins of the GABARAP family are processed by a ubiquitin-type conjugation machinery. structural investigation of complexes with its interaction partners has just commenced. The biological significance of the clathrinGABARAP interaction might relate to the GABAA receptor: endocytosis of the receptor is crucial for control of receptor numbers at the postsynaptic membrane of neurons. If this binding site is biologically relevant.Structural characterization of GABARAP-ligand interactions Y. Thielmann1 . W11 ) and a leucine side chain (L9 ). GABARAP interaction partners should display a surface-exposed conserved tryptophan. Among the GABARAP binding peptides identified by phage display screening.H. Therefore. GABARAP has been identified as a protein interacting with a cytoplasmic loop of the GABAA receptor γ 2 subunit. as discussed below. such an invariant tryptophan can be found in the binding motifs of the GABAA receptor γ 2 subunit as well as additional physiological ligands. Weiergräber2 . This process depends on the for- 102 . which are attached to the convex face of the β -sheet. It undergoes a C-terminal lipidation process that enables anchoring in the cytosolic leaflet of cellular membranes. It is important to note that ligand binding to GABARAP induces significant conformational changes in the protein. Willbold1 1 2 ISB-3: Structural Biochemistry ISB-2: Molecular Biophysics The GABAA receptor-associated protein (GABARAP) plays an important role in intracellular trafficking of several proteins. FIG. D. Although the K1 peptide makes contact with GABARAP in its entire length (Fig. In1: Complex of GABARAP with a high-affinity ligand (K1). O. Current evidence indicates that it might play a major role in subcellular transport of various cytosolic and membrane-associated proteins. with the final step involving covalent linkage to membrane lipids instead of proteins. GABARAP is depicted as ribbon model with the β -grasp domain and the N-terminal extension coloured in shades of blue. Colocalization with the GABAA receptor in cultured cortical neurons and the presence of a tubulin binding site qualified this protein as a potential adaptor linking the GABAA receptor to the cytoskeleton. complex formation appears to be dominated by hydrophobic interactions mediated by two tryptophan residues (W6 . GABARAP and its paralogues are members of the ubiquitin superfamily which is characterized by a so-called β -grasp fold. NMR titration of GABARAP with a 13-mer clathrin peptide revealed changes which are clearly indicative of a direct interaction. The number of known GABARAP interaction partners is rising steadily. Originally. Phage display screening for GABARAP binding peptides indicated a high prevalence of tryptophan residues in potential ligands. A hallmark of the GABARAP family is an N-terminal extension containing two additional α-helices. J. The peptide is shown with red and gray backbone. The specific behaviour of individual resonances indicated the presence of two apolar grooves on the surface of GABARAP which display different affinities for indole derivatives. such as calreticulin and the heavy chain of clathrin. The latter consists of a central four-stranded mixed β -sheet with two αhelices located on its concave side. Our recent work has established that GABARAP features two hydrophobic binding sites (hp1 and hp2). 1). the heavy chain of clathrin was identified as a physiological GABARAP binding partner. the "K1" sequence (DATYTWEHLAWP) was found numerous times in independent experiments. Mohrlüder and D. [4] Y.-D. Calreticulin is another GABARAP binding partner identified by a database search and verified using biochemical techniques [3]. a hexameric ATPase belonging to the AAA (ATPases associated with various cellular activities) class. The interaction with clathrin heavy chain may therefore represent an important aspect of GABARAP function in GABAA receptor trafficking. but the adjacent domains of calreticulin account for additional contacts. respectively.Proposed structure of the GABARAP-calreticulin complex. biochemistry and structural biology. Surface plasmon resonance experiments with calreticulin and GABARAP yielded a dissociation constant of 64 nM. FIG. FEBS J. Weiergräber and D. K. GABARAP has been identified as an interaction partner of NSF. Thielmann. FIG. T. T. R. Stangler. 5543– 5555 (2007). See text for details. H. Proteins (in revision). The ligand attains an elongated conformation in close contact to GABARAP. Thielmann. which ultimately govern the biological functions of this novel class of adaptor molecules. the core binding motif (CRT178-188 ) in blue. Mol. J. applying a wide repertoire of methods from molecular and cellular biology. Mohrlüder. The minimal machinery required to maintain membrane fusion in vivo includes NSF. H. 3. Mohrlüder. which is flanked by polar contacts. Y. our data have provided novel insight into structural foundations and dynamic properties of protein-protein interactions of GABARAP and its homologues. Stangler. this is the strongest interaction reported thus far for either protein. 1320–1331 (2008). Weiergräber. Hartmann. Wiesehan. 276. suggesting a regulatory impact on ATP binding and/or hydrolysis. Thielmann. Mohrlüder. 103 . NSF subunits are shown in blue and light grey. Wiesehan and D. using the related ATPase p97/VCP as a template [5]. [3] J. 274. FEBS J. Biol. Y. T. [2] O. O. GABARAP is displayed as surface representation. Docking experiments with GABARAP Model of hexameric NSF with docked GABARAP molecules. Becker. J. J. H. J. Whereas crystal structures have been determined for single domains of the NSF protomer. B. W. 3: resulted in the model shown in Fig. Together. Globular domain and P domain of calreticulin are shown in light and dark red. A structure model of full-length calreticulin reveals that its proposed GABARAP binding site is located at the N-terminal junction between the globular domain and the P domain (Fig. Willbold. In a subsequent study. with colours indicating residues affected in NMR titrations with CRT178-188 (blue) and the entire P domain (red). The proposed NSF-GABARAP interface is composed of an apolar core. O. Thielmann. 1767–1775 (2008). Mataruga and D. [1] Y. Weiergräber. K. Willbold. Willbold. NMR spectra of GABARAP in the presence of calreticulin showed clear indications of complex formation. These included the complete proline-rich (P) domain (aa 177-288) and a short segment thereof (aa 178-188) containing the putative GABARAP binding motif [4]. An interesting aspect of the complex model is the proximity of GABARAP to the nucleotide binding site in the major ATPase domain of NSF. A. Willbold. ChemBioChem 9. different calreticulin fragments were investigated for their interaction with GABARAP. 2: mation of coated pits and is mediated by clathrin. The three-dimensional structure of the GABARAP-CRT178−188 complex could be determined by X-ray crystallography. binding of GABARAP to NSF in this configuration could serve to anchor the complex to membranes. a structure of the full-length polypeptide is still lacking. K. These data suggest that the peptide contains the core binding motif. [5] Y. 1140–1152 (2009). H. Therefore. Due to its C-terminal lipidation. Willbold. These investigations have focused on the characterization of GABARAP interactions with artificial as well as physiological ligands. 2). Koenig. Hoffmann. Höltje and D. GABARAP in orange. interacting with both hp1 and hp2. Stangler. a homology model of the entire NSF complex was generated. which modulates the subcellular localization of the ATPase. 381. 104 . we have identified two domains where our expertise in nanotechnology can add value for improving the efficiency and sustainability of current environmental research and health care. Technological approaches also can be inspired by biological systems potentially leading to new cognitive and sensory approaches to information processing. but also paves the way to using it in derived sensory devices. Future in-vivo applications of bioelectronic devices include. and restoring damaged nervous pathways. and public security. Functional interfaces between neurons and micro-/nanodevices will have the potential to enhance in-vitro applications ranging from basic neuroscience research and disease modelling to drug screening and biosensors. such as biosensors.IBN-2: Bioelectronics Director: Prof. and ionic conductivity at radio frequencies. Within the broad field of bioelectronics.and millimetre wave range. Microwave to terahertz sensing techniques. for example. Ultra-sensitive superconducting quantum interference devices (SQUIDs) have a great potential for biomedical sensing. and information processing. 105 . redox proteins. Living cells and tissues exhibit an extraordinary range of functions including highly selective biochemical sensing (even in chemically noisy environments). Our research aims to develop bioelectronic devices that combine biological systems-from single biomolecules to living cells and organisms-with electronics. based on collectively vibrational modes of complex molecules at terahertz frequencies. managing pain. Bioelectronic devices and biomedical applications The use of biomolecules as the building blocks of higher-level functional devices will lead to applications ranging from the integration of biomaterials with electronics in recognition to sensing devices. airline. protein synthesis. enzymes and cells not only allows us to learn about molecular processes in biology. stimulating and recording deep-brain activity. thereby offering the potential to increase integration in combination with additional functionalities at the nanometer level. relaxation of the dielectric function in the micro. Sensing and imaging Electronic devices that detect trace amounts of biochemicals in the environment or in bodily fluids will allow far earlier detection than current technologies and will therefore facilitate appropriate reactions. medicine. Some of these have already had a profound impact on clinical diagnostics. Andreas Offenhäusser The interfacing of man-made electronics with bio-systems like DNA. Magnetic sensors have evolved due to the ever-increasing need for improved sensitivity. Bioelectronics research also exploits the use of biomolecules to perform electronic functions that semiconductor devices currently perform. have great potential for applications in biology. the ionic charge densities. A) Voltage+ clamp stimulation pulse. It can be directly correlated to the membrane current into the cleft. Here. the ions have to pass the cleft before entering the bath.2]. Due to the connected electric field other charged ions will move: ions with similar charge leave the cleft and move into the surrounding bath while ions of opposite charge are attracted and enter the cleft. the region between cell and sensor surface. The change of charge inside the cleft influences the source drain current under the gate and is sensed by the FET. Sommerhage1. Smaller letter-size corresponds + to smaller a concentration of K . metal microeclectrodes or field-effect transistors allows the recording of an extracellular component of these signals. The cell is approximated by a half-sphere with a radius a = 15 µm. The cleft height is h < 100 nm [1. While in the upper part of the cell (free membrane) these ions just enter the surrounding electrolyte bath directly. and the seal resistance. The distribution and strength of the potential near the sensor depends on the geometry of the cleft between attached cell and sensor surface and the specific electrical behavior of the cell membrane. The electrical activity of biological cells in vitro can be recorded by measuring changes in the local extracellular voltage with planar metal electrodes or integrated planar field effect transistors (FETs).2. it is different at the attached membrane. The results provide a method for determining the distance h between sensor surface and cell membrane. the electrodiffusion of ions in this interface is modeled by using the stationary Poisson-Nernst-Planck equations. Ingebrandt1. Biological cells (here: human embryonic kidney (HEK293) cells expressing a voltage gated K+ channel) are cultured on top of the gate of a FET [1]. 1.A Model for the Cell-Sensor Interface and Comparison with Experiment M. S. B) transmembrane K current measured by patch-clamp.2. Pabst1. F. We solve the equations analytically. FIG.2 1 2 IBN-2: Bioelectronics JARA-FIT Electrogenic cells are able to generate electrical signals which can be measured by various invasive electrophysiological methods such as patch-clamp or sharp microelectrode recordings. The flux of ions into the cleft causes an increase of charge. and derive expressions for the potential. 106 . and calcium ions (Ca2+). g. and C) corresponding ΔVFET recording. For an understanding of such extracellular signals it is mandatory to get detailed topographical as well as electrical information about the cell-sensor interface. Offenhäusser1. The cell is located on the top of a FET gate. Comparison with transmissions electron microscopy studies shows reasonable agreement. For a correct description of the signals. A. there is extracellular electrolyte solution. 2: Exemplary electrical coupling of a HEK293 cells + transfected with K channels to a FET gate. Outside the cell and inside the cleft. A schematic picture of a typical setup is shown in Fig. the ion channels in the cell’s membrane open and ions can flow from the inside to the outside. By electrical excitation. potassium (K+). The gate area of 2 the FET is about 50 µm . this interface can be described by a flat disk between cell membrane and sensor surface. In a first approximation. G. we receive h ≈ 70 nm.2. Growing cells on the surfaces of e. These changes in local extracellular voltage result from the flux of ions across the membrane of the cell.2. Wrobel1. mainly sodium (Na+). For human embryonic kidney cells. For a quantitative understanding of the extracellular signals it is necessary to describe the experimental situation. FIG. 1: Schematic drawing of the cell-sensor interface between a HEK293 and a field-effect transistor (not to scale). By correlating ΔVFET as a function of the total cell current IM in steady state case for many cells. DK. Ingebrandt. a differential equation system consisting out of the continuity equation. Baumann. Phys. the system is reduced to an ordinary inhomogeneous Bessel differential equation.1). (ii) an increase of VFET to a steady-state amplitude. Interf. 89. Wrobel. the measured average height h was in the range 35 – 40 nm. F. The FET signal shape resembles a combination of different signal components. Ingebrandt. Seifert. Wrobel. T. the last two formulae allow the calculation of an average height h. which is about half of the theoretical value. RJ = q B k BT 2 B 8πe0 ntot hDK with q. Offenhäusser. As a result we receive for the potential ψ(r) ψ (r ) ∝ ⎜ ⎜1 − ⎝ ⎛ r2 ⎞ L2 ⎟+4 D 2 ⎟ a ⎠ a2 ⎛ I 0 ( r / LD ) ⎞ ⎜ ⎜ I ( a / L ) − 1⎟ ⎟ D ⎝ 0 ⎠ + solution. The system describes the potential and charge densities inside the cleft.and nchannel FETs.Fig. that is absent in IM. it was found for HEK293 cells. 2): To compare our theoretical result for the height h. 5 (2008). A. – Soft Mat. kB. M. Eur. n tot. and (iv) a slow relaxation of VFET. 3: Comparison of the steady-state p-channel FET signals (ΔVFET) in dependence on the current through the attached membrane (IJM) for 10 cells. This equation can be solved analytically in the stationary case corresponding to the steady state pulse situation after 100 ms in Fig. Transmission electron microscopy study of the cellsensor interface. A. S. 1-8 (2007) [3] G. the Nernst-Planck equation and the Poisson equation [2]. ion density of the surrounding electrolyte 107 . Höller. diffusion constant of K ions and the height h of the cleft (Fig. G. In our approach we apply the Poisson-Nernst-Planck (PNP) equation system. By adapting the PNP system to our experimental situation. In presence of polylysine. each with distinct kinetics depending on the respective cell type: (i) capacitive transients. U. Here r is is the radial position inside the cleft. J. Wrobel. which means half of the signal comes from the gate properties and the other half from the membrane current into the cleft. H. 2 shows the comparison of whole-cell membrane currents (IM) and extracellular signal shapes (VFET) recorded with a p-channel FET for + HEK293 cells transfected with a K channel: (A) Rectangular stimulation pulses from the holding voltage led to (B) whole-cell current IM and (C) corresponding FET signals VFET for activation of the + K channel. 3628-3638 (2005) [2] M. caused by the capacitive coupling of the stimulation pulse. R. Sommerhage. F. which corresponds to a constant total charge density inside the cleft. A. Enderlein. S. temperature. It turns out that δ is of the order of 1. Ingebrandt. Ecken. Offenhäusser. E. J. While LD/a is of the order 10 . this is a reasonable agreement. (iii) a partially instantaneously decline of VFET at the end the pulse. the Boltzmann constant. The studies were complemented by imaging ellipsometry. S. Roy. LD the Debye-length and I0 the Bessel -5 function. Solution of the Poisson-Nerst-Planck equations in the cellsubstrate interface. From Fig. Considering the experimental uncertainties for growing cells on substrate. Bochem. The correction term δ takes into account the surface properties of the metal gate of the FET underneath the cell. B. J. P.57 MΩ and in consequence 70 nm for the height h. Soc. S. Dieluweit. that the contact geometry between cell membrane and substrate was dependent on the protein coating used for the substrate. the second term can practically neglected giving a parabolic potential. 3 we receive for RJ = 0. A. 24. With the above results an expression for the measured voltage signal ΔVFET of the FET in the steady state case can be derived (Fig. a the radius. e0. As a result. Biophys. From the potential the charge densities can be derived showing a similar parabolic form like the potential. Kaupp. H. Pabst. 2. elementary charge. Cell-transistor coupling: Investigation of potassium currents recorded with p. Transmission electron microscopy (TEM) was applied to analyze the cellsensor interface. FIG. Offenhäusser. J. h being a correction factor close to 1 due to the experimental situation. ΔVFET = (1 + δ )RJ I JM with the ‘cleft resistance’ RJ and the ion current IJM of the cell into the cleft. experiments have been made to measure the height directly [3]. Sommerhage. For the description of the FET signal shapes the ion flux in the cleft has to be understood. RJ is given by [1] G. F.2. Methods based on self organization can be applied on a larger scale but often lack the versatility obtained by the direct methods. 108 . at different stages of KOH etching (b. Furthermore. Mourzina1. Here. Interfaces with nanoscopic sizes play a vital role in electrochemistry. Nanopatterned surfaces promise to enhance the interface quality regarding to electronic signal transduction.d) directly after RIE (a). While the direct lithographic methods offer the most versatility they are usually limited to small area patterning due to speed limitations caused by the sequential processing. and template-assisted methods. Sommerhage1.Nanostructured Interfaces for Bioelectronic Systems B. FIG. energy storage/conversion devices. Y. and after removal of the silicon oxide mask (d). most fabrication methods based on self organization without template assistance are limited to specific materials. We therefore combined an imprint approach with a nanotemplate-assisted electrodeposition to create large-scale micro. cell adhesion. bioelectronics.b) and line patterns (c. 1: Process sketch for the fabrication of the largescale patterned arrays of Au-nanopillars.2. Offenhäusser1. Imprint techniques are often preferred over other direct patterning techniques. we present a concept of a large-scale patterned gold nanopillar array interface on semiconductor substrates with the aim of functional coupling to electrogenic cells. cell adhesion studies. D. This new and versatile method can be adopted for different interface materials of electronic circuits and their coupling to both cellular and molecular sensing components.and nanometer sized arrays of gold nanopillars at predefined positions on a semiconductor substrate. Hofmann1. Brüggemann1. Electrode materials with increased surface area have the best electrode properties due to a large effective area. catalysis. Thereby. Wolfrum1. and information technology. This reduces the noise level and increases the current injection capability. nanoparticle growth or deposition by self organization. a larger capacitance. FIG. because lithography has to be used only for the fabrication of the stamps.2. 2: SEM images of silicon stamps for imprinting aluminum layers (a-d). Fabrication methods of NPAs can be divided into direct lithographic methods utilizing e-beam or focused ion beam (FIB) writing. Top and side view of dotted “FZJ” (a. A. Intensive research is focused on patterned nanopillar (NPA) array interfaces of diverse materials.2. B. The line width at the stamp tip is 130 nm. Nanostructuring is based on the combination of an imprint approach with a nanotemplate-assisted electrodeposition. and selectivity.c).2.2 1 2 IBN-2: Bioelectronics JARA-FIT The properties of a bioelectronic interface (interface between an individual cell and a chip transducer) are of primary importance for bioelectronic systems. and further decreasing of the feature size. the specific and precise positioning of nanofeatures on a large scale is especially challenging. favouring a smaller electrode resistance. The inset shows an image enlargement of a single dot feature of (b). Offenhäusser. Smaller interspace distances might be achieved by performing multiple imprints with the same stamp using a precise positioning system. Similar arrays could be used for surface enhanced Raman scattering [3] and cell adhesion studies [4]. 3b). which possibly remains at the bottom of the pores originat ing from the predefined regions. The template material can be selectively etched in alkaline solution to expose the gold nanopillars arrays (d). in which gold is exclusively formed inside the nanochannels of the porous alumina. Kessler. is dissolved in 5% phosphoric acid. Y. Schwaab. Each nanopillar array is composed of approximately 60 Au-pillars with a width and height of ~35 and ~300 nm. B. Mourzina. Images (c-f) show top and side views of Au nanopillar fences created by using line stamps to indent the aluminum film. Schaal.-P. Small 2 (2006) 1256. Nano Lett.and mircopatterned regions of pores on a substrate. In Fig. Phys. B. Fox. M. 024308. Fig. Selhuber. 4a. Patterned aluminum films are subsequently anodized in 0. S. Fig. Schwaab. The lines extend up to the stamp size of 4 mm. The minimum interspace distance of the patterned nanopillar lines is determined by the resolution of the optical lithography used for the stamp fabrication. We propose that this approach can be extended to different interface materials of elec tronic circuits and their coupling to both cellular and molecular sensing components. Y. Here. and cover an area of 4x4 mm² on an 11x11 mm² chip. 4b. Fig. Mayer. Wolfrum. The stamp patterns have different geometries. [1] B. F. Hermanns. We further showed that the nanotemplate-assisted electrodeposition can be combined with a prepatterned electrode array as a precursor for NP growth to create a new type of a 3-D nanostructured interface for coupling with electrogenic cells. 6 (2006) 398. S. Dr. The barrier layer. J. J. Müller. Lepsa. Sommerhage. the Au-nanopillars depend on anodization conditions of the aluminum film and the subsequent pore enlargement in phosphoric acid. For example. we fabricated nanopillar arrays with a diameter down to 20 nm and a pillar to pillar distance of ~50 nm by anodizing the aluminum film at 22 V in sulfuric acid (3%). Figure 3(c-f) shows images of patterned Au-nanopillar line arrays. [2] [3] [4] 109 . Offenhäusser. 3a) exhibiting dots of different interspaces can be reproduced with pads of gold nanopillar arrays (Fig. Nonn. H. respectively. Acknowledgements: A. Mourzina. we show that an imprint approach on thin aluminum films can be used to define nano. H. Nano Letters 6 (2006) 453.The process for the fabrication of patterned nanopillar arrays is shown in Fig. 2. Bochem. G. D.. D. D. Ingebrandt. which selectively act as a template for the electrodeposition of nanopillars. Prömpers. The pattern of the stamp is transferred via imprinting onto a Ti/Au/Al covered substrate (b). A. Steffen. C. Appl. the smallest initial feature size of the stamp being 1 µm. AM acts as a template material for the metal deposition. N. Wolfrum. 2005.3 M oxalic acid at 3°C and 40 V to obtain nanoporous alumina membrane (AM) template with an approximate interpore distance of 80 nm (c) [2]. D. P. Sauer et al. A. J. 1. FIG. Dr. Dr. D. The interspace distance and width of We found that the nanostructured surfaces presented here were biocompatible with cell lines expressing ion channels as well as primary neuronal cells from the rat cortex. Spatz. M. Walter. FIG. 97. The Si stamps for the indentation of the aluminum surface (a) are created by lithography combined with anisotropic wet chemical etching of silicon [1]. A. Mayer. 4: SEM image of a rat neuronal cell growing on a gold nanopillar surface (a) and a microelectrode active area modified with gold nanopillar pattern (b). The anodization is stopped after the nanochannels originating from the indented surface reach the underlying gold substrate. M. 3 it is shown how a large-area stamp pattern (Fig. 3: FZJ-silicon stamp pattern (a) and the reproduction of the stamp pattern as Au nanopillar arrays (b). Functional Networks of Rat Neurons on in-situ Patterned KDI S. Meffert1,2, K. Adamiak3, R. Helpenstein1,2, B. Hofmann1,2, A. Offenhäusser1,2 1 2 IBN-2: Bioelectronics JARA-FIT 3 DWI, RWTH Aachen University This study aimed to increase the lifetime and stability of in vitro network of neurons. We tested KDI, a 12 amino acid peptide of γ laminin peptide, using in situ-microcontact printing to create a chemically attached peptide pattern on silicon oxide substrate. By atomic force microscopy (AFM) a pattern thickness of about 2.9 nm which corresponds to a peptide monolayer could be measured. Rat cortical neurons were seeded on the KDI pattern and tested on their viability and functionality. Synaptically connected neurons were confirmed by double patch-clamp recording and immunostaining experiments. The expression of the pre- and postsynaptic proteins vesicular GABA transporter (VGAT) and gephrin could also be evidenced. Our data indicated that a monolayer of in situ-printed KDI peptide is sufficient for neurons adhesion and also for formation of functional and stabilized networks. By future application of these patterns as growth substrate on sensor arrays the extracellular recording of single neurons should be further improved. There is an increasing need in the field of neuroelectronic devices to immobilize functional proteins on the device surfaces for coupling extracellular signals from neuronal networks and long-term recording. Immobilization on surfaces can be easily accomplished by direct adsorption. However this method results partially thick and inhomogenous protein layers, denaturation of the protein, as well as unstable attachment affecting neurons and network function. Covalent attachment of functional proteins or peptides are one solution and are important when a coated substrate is subjected to a flowing solution or exposed for a long period of time in solution. In this study we aimed to construct a 1) chemically stable, 2) reproducible, 3) thin as possible growth pattern enabling long time stability and functionality of neuronal networks on silicon oxide substrates. Thereby, we used the in situmicrocontact printing (µCP) which was recently established to print proteins/peptides without inducing their denaturation [1]. In situ µCP was combined with a protocol for covalently attaching peptides to construct a chemically stable peptide pattern of KDI, a 12 AS consisting synthetic peptide deriving from neurite outgrowth-promoting domain of γ laminin. KDI (cystein-terminated KDI peptide, 5 µM) was printed on aminosilanized (APTES, 3-aminopropyl-trietoxy silane) and crosslinker (sulfo-γ-maleimidobutyryloxysuccimide ester) attached glass substrate. To investigate the fidelity of the pattern, carboxyfluorescein labelledKDI was used for one set of experiments. Printed KDI patterns were first analyzed by phase contrast and fluorescence microscopy. Fluorescence images confirmed the transfer of carboxyfluorescein labelled KDI on glass substrate and illustrated that the pattern was almost without any interruptions and a high degree of uniformity. FIG. 1: AFM analysis was performed using Nanoscope IV Multimode Instrument with a Nanoscope IV controller and a 15 µm scanner in tapping mode. The KDI in-situ imprint shows a smooth topography of the printed pattern (A) and a section analysis with a small roughness of about 1nm (A, right). Detailed analysis (B) revealed a layer thickness of 2.868 nm (B, right). 110 In contrast, no KDI pattern was observable by phase-contrast microscopy which indicates a reduced thickness of this peptide pattern in comparison to the standardly printed protein mix PECM (polylysine/ extracellular matrix gel) [2, 3]. For further characterization of the peptide imprints and measuring the layer thickness atomic force microscopy (AFM) was carried out using a Nanoscope IV Multimode Instrument with a Nanoscope iV controller and a 15 µm scanner. Samples were scanned in tapping mode with resonance frequencies between 260 and 300 kHz. The KDI-imprint showed a smooth topography of the pattern indicating a homogenous peptide coupling to the surface. Further AFM analysis of one section confirmed the slight layer roughness and revealed a thickness of 2.9 nm (Fig.1) which was in the range of the theoretical calculation of a peptide monolayer [4-5]. The KDI pattern was subsequently tested in cell culture on neurons functionality and network stability. guidance of the processes along the lines were achieved with high compliance. The neurons developed a mature morphology and differentiated to network forming neurons. Apart from the morphological evidence the neuronal identity and functionality was confirmed by immunostainings and electrophysiological experiments. Single and double patch clamp experiments elicted spiking neurons and synaptically connected neurons grown on the KDI pattern. The cortical neurons were stained with antibodies against the pre- and postsynaptic proteins, vesicular GABA transporter (VGAT) and gephrin. The expression of both proteins could be identified at DIV 7 and DIV 12. Finally, the network formed on KDI pattern were stable without detaching from the substrate up to DIV 22. In summary, we were able to construct a chemically stable, homogenous, 2.9 nm-thin KDI and likely monomolecular peptide layer. The peptide layer was tested to be suitable as substrate for adhesion and outgrowing of neurons confined to the pattern geometry. This peptide pattern could further be proved to be suitable for long-time culturing of neurons and due to the stability of networks in cell culture. With these data, we propose that KDI patterned substrate may be useful for extracellular recordings to improve the signal coupling by reducing the celldevice distance. We thank D. Mayer and S. Gilles for AFM measurements. Financial support came from the Sony Deutschland GmbH. [1] D. Schwaab, Dissertation at the RWTH Aachen (2007). [2] A. Vogt, L. Lauer, W. Knoll, A. Offenhäusser, Biotechnol. Prog. 19, 1562 (2003) FIG. 2: Phase contrast image of rat cortical neurons grown on KDI pattern DIV 7, forming geometrically defined networks (A). Patterned neurons were fixed and immunostained with antibodies against the pre- and postsynaptic proteins VGAT (red) and gephyrin (green). Laser scanning images of neurons grown on KDI immunostained against VGAT and gephrin showing high expression of both synaptic proteins DIV 7 (B) and DIV 12 (C). [3] S. Böcker-Meffert, T. Decker, S. Schäfer, A. Offenhäusser, PB-10, 5th Int. Symp. Biomimetic Materials Processing (BMMP-5), 2005 [4] M. Scholl, C. Sprössler, M. Denyer, M. Krause, K. Nakajima, A. Maelicke, W. Knoll, A. Offenhäusser, J. Neurosci. Methods 104, 65 (2000) [5] K. Adamiak, B. Hofmann, S. Böcker-Meffert, A. Offenhäusser, 7th Göttingen Meeting oft he German Neurosciences Society, Germany, 738-5B (2007). Cortical neurons from embryonic (E18) rats were prepared in a chemically controlled medium and seeded (16.000 cells/ cm2) [2] at the KDI patterned glass substrate. The substrates were optically controlled due to adhesion and outgrowth of the cells on the pattern. Figure 2 illustrates the behaviour of the neurons after 7 and 12 days in vitro (DIV7). The aligning of the cell bodies onto the 12µm-nodes of the grid pattern as well as the 111 Generation of protein gradients by nanoscale patterning D. Schwaab1,2, P. Zentis1,2, S. Völker1,2, S. Meffert1,2, D. Mayer1,2, A. Offenhäusser1,2 1 2 IBN-2: Bioelectronics JARA-FIT Combining high resolution lithography with microcontact-printing by means of hard plastomers it was possible to adjust the size of elements of a protein pattern and simultaneously the distance between them with sub 100 nm resolution. Rat neurons adhered onto these sub 10nm protein layers, apparently integrate over a large number of pattern elements and recognise the pattern as quasi homogeneous. However, the neurites showed pre-dominantly an aligned outgrowth corresponding to the underlying pattern. Thus, the technique proposed in this work maybe path the way to systemically study the influence of nanometer sized purely biochemical gradients on guiding neurons. These discontinuous gradients will further enable to evaluate the role of ECM/ligand spacing and in parallel the effect of defined gradient parameters on axon guiding. In developing nervous system the path of axons to reach their targets and establish neural circuits is directed by soluble and surface-bound biochemical gradients. Ligands of these gradients are detected by axonal growth cones which probe their environment by extending or retracting their filopodia. Although considerable effort has been directed towards characterizing chemotactic molecules and their receptors, the cellular mechanism by which neuronal growth cones sense these gradients remains generally unknown. It is envisioned to fabricate chemical gradients with length scales relevant to the biological involved cellular structures. The fabrication of continuous surface-bound gradients has been realized by a wide variety of methods [1]. Recently, von Phillipsborn et al. [2] used microcontact-printing to produce geometrically defined discrete gradients consisting of protein-covered spots varying in sizes and spacing. To transfer the concept of discrete gradients to the nanoscale dimensions of extracellular matrix fibrils nanostructuring techniques needs to be adapted. In the scope of this work we have developed a procedure to fabricate discrete purely biochemical gradients of extracellular matrix proteins by means of microcontact printing (µCP). This technique directly transfers molecular inks from a polymer stamp to the target surface [3]. Here we show the transfer of ultra thin (10 nm or less) compact protein layers with sub 100 nm lateral resolution and demonstrate the use of these films to guide neuronal outgrowth. Polyolefine plastomers (POP) were chosen as stamp material with a Youngs modulus of E=80 MPa (Avinity VP, Ticona). The high stiffness prevents the material from disrupting effects like sagging during printing process and facilitates high pattern fidelity. The plastomer stamps were structured by hot embossing by using a mold made of silicon. The molds contained a pattern with lines and spaces with both constant and varying widths ranging from 1 µm to 75 nm, resulting in a line pattern with different pitches. A mixture of FITCpoly-D-lysine extracellular matrix proteins and (FITC-PDL/ECM) was used as ink. The stamps were dried with a nitrogen stream and pressed onto glass or silicon oxide substrate which was prehydrophilized by oxygen plasma. An uniform transfer of proteins on large length scales was verified by optical fluorescence microscopy (Fig. 1a), indicating also a high pattern fidelity. Images recorded at higher magnification by means of scanning electron microscopy (SEM) did not reveal any major pattern defects. Well-defined protein lines were observed as dark stripes in the SEM images (Fig.1b) with inverted contrast compared to fluorescence images. In the SEM images neither hints of disruptive effects like sagging or pattern collapse nor contrast variation over the imaged area were observed. FIG. 1: Large scale images of the transfer of FITCPDL/ ECM ink to a SiO2. (A) Fluorescence microscopy image of the FITC dye coupled to PDL (excitation 488nm, emission 518nm) indicates schematically the area of the SEM image, see right. (B) SEM image at a magnification of 5450 with inverted contrast compared to (A), the dark 112 PNAS 2002. On the patterned surface. These discontinuous gradients will further enables to evaluate the role of ECM ligand spacing and in parallel also the effect of defined gradient parameters on axon guiding. Nelles. Bastmeyer. Yasuda. V. which results in a rather poor contrast and small protein features are difficult to reveal at some places. F. S. FIG.The homogeneity of the protein coverage was further investigated with nanoscale resolution using Atomic Force Microscopy (AFM). P. 2004. After 5-7 days in culture the cells were fixed. S. there was no obvious difference in the number of cells adhered on nanopatterned area (64 ± 5) compared to the area of the surface that were homogeneously covered with proteins 124 ± 29. 1. Offenhäusser. The AFM images revealed that all structural elements of the pattern were transferred from the stamp to the hydrophilized SiO2 surface. Best. The cells adhered to the homogeneously coated and patterned areas with no differences due to the fact that even the largest elements of the pattern (1 µm x 5 µm) were much smaller than 2 the adhesive area (120 µm ) of the soma size of cortical neurons. von Philipsborn. Li. On the left side of the image the nano pattern design was superimposed on the original SEM image to highlight that the filipodia attached also to the smallest elements of the pattern. clear differences were observed for the growth of neurites on the patterned area compared to the homogeneously coated one. T. A. Lang. Lehnert. P. J. Gasteier. 2487 [3] A. Neurosci. the SEM images revealed that the neurons extended neurites > 100 µm which also arborized on both particular areas of the substrate. Helpenstein. S. Normalized to the coverage with proteins. D. A. Figure 2 shows a SEM image of a SiO2 substrate after printing of FITC-PDL/ ECM and culturing cortical neurons for 7 days. Knoll. [2] A. X. A. R. The cells adhered to both areas however with distinct differences regarding neuronal outgrowth (data not shown). Soft Matter.2 kV. The height of printed protein layers varied between 2 nm and 10 nm. The isolated neurons were obtained from 18 day-old rat embryos (E 18) as described by Vogt et al. M. The cells were plated onto patterned SiO2 substrates. G. Development 2006. W. C. Therefore. G. Furthermore. A. J. the designed pattern was superimposed onto the SEM images aligned to the protein pattern underneath in order to highlight the areas which are covered with proteins. 134. [4]. Jiang. [1] K. Averaged over the whole pattern the coverage of the surface with proteins was estimated to 45 % which is consistent with the particular pattern design. Loeschinger. Schulte. Z. Vogt-Eisele. Reska. Bonhoeffer. Imaging parameter were selected such (voltage 0. Vogt. Groll. To evaluate the response of neuronal cells to the nanoscale discrete protein gradients described above we analyzed the adhesion and outgrowth of primary neurons on these patterns by SEM. The SEM images further confirmed the alignment of the neurite growth with the pattern. 3. 2: SEM image of a patterend SiO2 substrate with outgrown neurites of rat neurons. Böcker-Meffert. However the shallow protein layers (2 – 10 nm) were buried underneath the sputtered iridium layer. even the 75 nm lines. In contrast. even though a few neurites do not follow the underlying pattern. the technique proposed in this work may path the way to systemically study the influence of nanometer sized purely biochemical gradients on guiding neuronal cells by directing neurite outgrowth. Bernard. Dertinger. Growth cones could also be imaged demonstrating that the filipodia adhere at different structure elements including the smallest ones with a width of 75 nm. critical point dried and coated with a thin layer of platin/iridium. 113 . Stefani. David. 133. Schäfer. Offenhäusser. Möller. Decker. F. On the homogeneously coated surface the neurites formed an extensive meshwork of stochastically criss-crossing neurites. For a better analysis of the effect of nanopatterns on the neurite growth SEM images with a higher magnification were recorded. 290 [4] A. A. 12542. Murthy. 2007. A. inlens detector) that both protein pattern and neurons with outgrown neurites are displayed simultaneously (Fig. 191. Thus. The image was recorded at the transition from a nanopatterned area to an area homogeneously covered with proteins. M. Whitesides. J. neurites showed predominantly an aligned outgrowth corresponding to the underlying pattern indicating that the structure elements of the gradient were recognized in both main directions of the nanopattern which are perpendicular to each other. The neurites grow in alignement to the pattern of FITC-PDL/ ECM. An analysis of the line width and the deviation between designed pattern and printed features for most structures was smaller 10 % in average. Meth.2). 114 . In this vast field we focus on the mesoscopic length scale from molecules to cells where principles of soft matter physics shape biological processes. Therefore researchers from all three classical sciences form the institute’s scientific staff. chemistry. At present. adhesion. molecular aggregates. locomotion. To achieve our goal. In order to pursue this double strategy we develop and apply advanced methods for measurements on living cells and biomolecules as well as preparation and analysis techniques for tailored biomimetic systems. and mechanosensing of living animal cells are at the center of our interest. Rudolf Merkel Our research is directed towards a basic understanding of the physical principles. On this length scale we study mechanical functions and properties of living cells. and biology.IBN-4: Biomechanics Director: Prof. and molecules. we perform complementary experiments on living cells and biomimetic model systems. Interdisciplinary cooperation on common projects on an everyday basis is one of the major strengths of the institute. underlying structure and function of living cells. force generation. By studying living cells we gain insight into the physiological relevance of processes and their likely mechanisms. experiments on model systems designed to mimic bioadhesion and the cytoskeleton enable us to achieve a quantitative understanding. 115 . Further. Our research strategy traverses the boundaries between physics. The characteristic smoothing length is proportional to the ratio between the surface tension and Young modulus of the material. Eindhoven . for very soft rubber E may be of order 10 kPa and if γ is of order 10 mJ/m2 we get D ≈ 1 µm. e. 70 µm width. 5. S. Care & Health Applications. 2H. 2H’perfluorooctyl-trichlorosilane (Sigma. 20. The mixtures were poured over silicon oxide molds patterned by conventional optical lithographic techniques. This can be understood from the following qualitative argument: the elastic energy stored in a solid object.5 µm squares and lattice constant of 3.J. 10. with linear size D and with uniform strain of order unity. as can be seen in Figure 1. and the whole ensemble was cured over night at 60 ◦ C. Louis. The experiments were performed using silicone rubber prepared using a two part kit. 1: (a. 48. Mayer3 . 17. In order to illustrate the strong elasticity effect. soft micrometer sized solid objects may undergo shape deformations corresponding to a strain of order unity.g. Merkel1 1 2 3 * IBN-4: Biomechanics IFF-1: Quantum Theory of Materials IBN-2: Bioelectronics now at Philips Research Laboratories. the silicon oxide masters FIG. very soft solids are affected by surface tension at scales relevant for micro-technology and biology. PDMS rubber with Young moduli of 11. The comparison between 10:1 and 50:1 can be seen in Figure 1. e) stiff 10:1 PDMS (1. Bo N. Thus. Two types of mold patterns were used: the first one consisted of a square lattice with 2. scales with the volume of the solid as ED3 where E is the elastic modulus. 144 kPa and 1.D. Persson2 .Deformation of Soft Solid Objects: Resolution Limit in Replica Molding O. R.6 MPa) and (c.M.The Netherlands The shape of liquid droplets results from the interplay between surface tension and external forces. Sylgard 184. St.* . A glass coverslip was placed over the PDMS layer. Hoffmann1 . In order to prevent PDMS sticking to the mold. C. Therefore nano and micromolding using elastic material will produce replicas with a characteristic smoothing length scale of γ/E .6 MPa was molded from mixtures of 55:1. In this case the “re- 116 . MO). The surface free energy scales with the surface area as γD2 . B. 1d). 30:1. The elasticity of the resulting rubber depends on the cross-link density via the mixing ratio of the two constituents. D. where γ is the surface free energy per unit area. 40:1. 50:1. Upon curing the mixture undergoes a cross-linking reaction which will produce a solid elastomer. Usually. This qualitative conclusion has many practical implications. We will assume that h0 << λ. By adjusting the mixing ratio of the base (vinyl terminated polydimethylsiloxane) to the curing agent (methylhydrosiloxane-dimethylsiloxane copolymer) in the range from 10:1 to 55:1 the elasticity of the resulting rubber can be tunned between 2 MPa to 10 kPa. stamps for micro-contact printing. for microtechnology and biology. f) soft 50:1 PDMS (17 kPa) replicas. 1a) and the second one had long trenches of 2. 50. Thus one may expect a strong shape deformation if D is of order γ/E [1]. were silanizated in vacuum using 1H. (b. Gordan1 . respectively. MI). 20. As an example. The PDMS surfaces were scanned in 1% Triton X-100. 93. Here it will be demonstrate how the competition between surface free energy and elastic (bulk) energy will modify the surface profile of a solid. In a similar way. with a 100 µm spacing in between (Fig. Cesa1. The shape change due to the surface tension and elasticity can be calculated starting from a sinusoidal surface profile with amplitude h0 and wavelength λ: u0 = h0 cos 2πx λ . 1H’. Dieluweit1 . micro-fluidic channels and micro-textured surfaces are produced using PDMS mixtures of 10:1 (base:to cross-linker) [2] giving accurate replicas at this length scale (micrometer). 35:1 and 10:1. Before the molding process the PDMS was degassed in a desiccator using a mechanical pump. purchased from Dow Corning (Midland. d) AFM images of SiO2 molds..5 µm (Fig. Appl. This is indeed visible in Figure 3 which shows the normalized height change for the 2 and 5 µm lines produced in different molds. equation (2) giving a good analytical expression to the rule of thumb ”the stiffer the better”.D. G. and 93 nm. where E ∗ = E/(1 − ν 2 ). ReV. Am. Westergaard. Theory curves were calculated using an interface tension of 8. The calculated profiles for the 2-. Whitesides. Mech. [1] O. Moreover. with E the Young modulus and ν the Poisson ratio. The surface energy is: 2 Es = γ d x 1+ du1 (x) dx 2 1 2 ≈ A0 γπ 2 2 h1 . B. Thus. 3: Normalized height change of the PDMS replicas produced in lines molds (1d) with depths of 531. C. Mayer. Mater. Error bars denote the standard deviation of the values measured at the different mold depths.5 mN/m and a stiffness of 11 kPa were considered in the calculation. and 20.N.J. 49 (1939) 117 . Trans. 28. Sci. 153 (1998) [3] H. Persson. 6. 5-. but the same wavelength. In general a surface with a variation in one direction can be mathematically described in the Fourier formalism as: h0 (x) = dqh0 (q )eiqx (3) with q = 2π/λ. Experimental results for line widths of 2 µm (triangles) and 5 µm (squares) are shown along with the theoretical results. Annu. Thus. 5-.laxed" surface profile u1 would still be sinusoidal.µm -wide lines compared with the experimentally measured ones are presented in Figure 2. Starting with a surface profile like the one in Figure 1e for h0 (x).Langmuir 24. Poisson ratio and the surface tension of the material. corresponding to the local pressure distribution [3] p= πE ∗ (h0 − h1 ) cos λ 2πx λ . J. 10-. 299.. R.M. 4λ FIG. Then the relaxed surface profile is h0 (x) = dq h0 (q )eiqx 1 + 2qγ/E ∗ (4) Even for stiff PDMS (≈ 2M P a) molding fidelity will become problematic at smaller length scales. S. Mech.5 mN/m.5 mN/m. Dieluweit. Cesa. (1) For surface profiles characterized by some width λ and some height h0 we expect the scaling ∆ h = h0 − h1 ∼ γ h0 E λ (2) FIG. the surface displacement in normal direction is u = u0 − u1 = (h0 − h1 )cos 2πx λ . 6636 (2008) [2] Y. with a reduced amplitude h1 . Gordan.µm -wide lines molds of 531 nm depth. Equation (4) can be used for exact surface topography calculation of the resulting replica molding samples when the elasticity and the surface tension of the material are known. where A0 is the nominal area of the surface. ∆h/h0 measured at the center of the line can be predicted well within the error bars of the AFM measurement knowing only the elasticity. B. Equation (2) indicates that the expected change in height is proportional to mold depth. Merkel . 10-. 2: Measured (solid lines) and calculated (dashed lines) height profiles of lines molded in 11 kPa PDMS using 2-. the elastic energy can be written as Eel = 1 2 d2 x u(x)p(x) = A0 πE ∗ (h0 − h1 )2 . and 20. with an interface tension of 8. Xia. An inverse approach would give a direct measure of the surface tension for soft solids in the case of known Young modulus and expected surface profile. λ2 Minimizing the total energy Eel + Es with respect to h1 gives h1 = h0 4πγ ≈ h0 1 − 1 + 4πγ/λE ∗ λE ∗ . As the AFM scan was done in Triton X-100 a surface tension of 8. Soc. Hoffmann. D. Eng. the surface profile from Figure 1f can be calculated using Equation (4). 184. For single FAs of WT cells. University of Bonn Presenilin 1 (PS) is a critical component of the γ-secretase complex that cleaves transmembrane proteins. Since earlier experiments indicated an influence of PS on ephrinB1 putatively affecting c-Src activity. acting as coactivator for c-Src transcription. Displacements of the regular micropattern were visualized by RICM and the generating forces were calculated from these data. Alternatively. The direct regulatory connection between PS1 and c-Src is ephrinB2. the number of FAs was reduced by 40%. WT cells were characterized by well visible FAs. analyses revealed forces in the range of 13 nN (Fig 1B) with a generalized first moment of -/about 8. Therefore. -/Very different results were found for PS1 cells. Cells were either analyzed using phase contrast or transfected with GFP-fusion proteins and analyzed by fluorescence microscopy. I.Y. To characterize the exact function of PS in cellmatrix adhesion processes wild-type (WT) and presenilin 1 knock out mouse embryonic fibroblasts were cultured and used for immunofluorescence analyses against actin and vinculin as marker of focal adhesion sites (Fig.65 µm² on average. B. PS1 deficiency was also associated with decreased tyrosine phosphorylation levels of FA specific proteins. Presenilin affects many mechanisms and one of these is the ephrinB/Eph receptor mediated cellcell adhesion. we -/found for PS1 cells an altered morphology with significantly reduced sizes of focal adhesion sites (FAs) compared to wild-type. n=86). a protein vitally involved in focal adhesion formation. cell forces were reduced by 50%. S. 1). Northern blot. Kirchgeßner1. Cell analyses were performed using wild-type mouse embryonic fibroblasts as well as PS1-/mutant strains. N. Here. which cleaves transmembrane proteins within their transmembrane domains leading to the release of the two cleavage products from the membrane (1). The freed products can play important roles in signalling pathways by translocating into the nucleus and acting as transcriptional coactivators (2). we conclude that γ-secretase is vital for controlling cell adhesion by transcriptional regulation of c-Src via ephrinB2 cleavage. J. a receptor mediating cell-cell adhesion. we identified a strong reduction in FA size upon PS1 deficiency associated with a reduction in cell force formation by over 50%. which was caused by a transcriptional down-regulation of c-Src kinase. Both effects went along with a decreased phosphorylation level of FA associated proteins and were caused by a PS1 dependent downregulation of c-Src on the level of transcription via ephrinB2 cleavage. called stress fibers. Since ephrins additionally bind to cellular sarcoma protein kinase (c-Src). 118 . Poisson’s number = 0. Furthermore. PS1 cells instead applied forces in the range of just 7 nN per focal adhesion with a generalized first moment of 3. Here. This process plays an essential role in signal transduction and vital functions such as cell adhesion. For a quantitative measurement of cell forces WT -/as well as the PS1 mutant strain were seeded on micropatterned. we analyzed the phospho-tyrosine levels of FAs in WT and PS1-/-. Merkel1. Niediek1.0 pNm (σ=3.1pNm. Waschbüsch1. Prominent actin stress fibers were absent. Cell biological procedures were performed according to standard protocols. Elastomeric substrates were prepared and calibrated (Young’s modulus = 13 kPa. n=36).Presenilin 1 Affects Focal Adhesions and Cell Forces via c-Src Regulation D. were connected. Here. Tamboli2. western blot as well as qRT-PCR experiments were performed according to standard protocols. Walter2. Born1. EphrinB2 becomes cleaved by PS1 with a subsequent translocation of the ephrinB2 intracellular domain (ICD) into the nucleus. moving phenotype. R. soft PDMS-substrates. Their spatial distribution changed from a cortical to a disperse localization. PS1 is an aspartyl protease forming the active components of the γ-secretase complex. it is speculated that PS might be an important regulator for switching cell function from a sessile to a dynamic.5) as described earlier (3).7 pNm. These sites with an average size of 0. V.9 µm² were mainly located at the cortex of cells and had an elongated shape. To every FA thick F-actin bundles. Hoffmann1 1 2 IBN-4: Biomechanics Department of Neurology.5 pNm (σ=4. cells were fixed and proteins were stained by immunolabelling. The average size of FAs was diminished by 30 to 40% and reached 0. 3B). Since two of them. 2B). were also known binding partners of c-Src. Cupers. expressed in WT and PS1-/. Since phosphorylation in FAs is mainly performed by activated c-Src kinase we analyzed the activation status of this kinase at tyr418 as well as -/its expression level in WT and PS1 cells. EphrinB2 cytoplasmatic domain is translocated in a PS1 dependent manner into the nucleus. 2 Crude protein (A) as well as total RNA (B) were -/isolated from WT and PS1 and analyzed for indicated proteins/mRNAs. Reduced levels of c-Src protein were therefore likely caused by transcriptional downregulation rather than increased protein degradation. Under-lying forces applied at every FA (red) as well as the generalized first moment (blue) were calculated as described in Cesa et al. we analyzed ephrinB2 in more detail. P.0 2 µm².cells and its localization was compared to GFP only. respectively. To complete the signal transduction pathway from PS1 function to c-Src activity. Fig. -/- [1] B. The experiments revealed c-Src regulation mainly at the transcriptional level. We therefore tested ephrinB2 intracellular domain (EB2-ICD) for transcriptional co-activator function. Instr. Ceşa. D.FAs was almost absent (data not shown). σ=0. An EB2ICD-GFP construct was expressed in WT as well as PS1-/. c-Src phosphorylated at tyr418 was decreased by 90% in the absence of PS1 compared to WT. -/- Life cell imaging revealed intense nuclear localization of EB2ICD-GFP (Fig. These analyses were performed in fixed cells using a phospho-tyrosine specific antibody and revealed high levels of phosphoryla-tion in FAs of WT cells while phosphorylation of PS1-/. Protein levels of c-Src were reduced by 60% in -/PS1 cells (Fig. W. n=100 FAs) (Fig. J Shioi. (1999) Nature 398. P. we checked γsecretase targets for a putative influence on c-Src. Georgakopoulos. Kirchgeßner.Fig.4 µm .cells. As given in Figure 2A. N. S. 3A). S. W. K. Northern analyses identified an almost identical reduction of c-Src transcripts. Note that translocation was only detected in the presence of functional PS1. 893-902 [3] C. 78.cells were also restored in tyrosine phosphorylation intensity and size (1. Schwarz. Saftig. M. Merkel (2007) Rev. Annaert. While nuclear localization could be observed for EB2-GFP in WT cells no such signal was present in PS1-/. levels of c-Src mRNA were reduced by 50% -/in PS1 cells (Fig. Cui et al. 1 (A) WT as well as PS1 cells were stained for actin and vinculin in immunolabelling experiments. U. a full length EB2-GFP construct. Sci. Hoffmann. De Strooper. Here. 3A). FAs of transfected PS1-/. identified the dependency of EB2-ICD translocation on PS1 function. 3 (A) PS1 cells were transfected with an EB2ICDGFP construct and analyzed in fixed cells for GFP (green) as well as tyrosine phosphorylation of FAs.(Fig. (1999) Mol Cell 4. As such result might have been caused by either regulation of autophosphorylation or regulation of expression. 518-522 [2] A. Craessaerts et al. α-tubulin and 28S rRNA. R. 2A). A similar increase was observed for c-Src in western analyses (not shown). (B) Cell types as in (A) were grown on elastomeric substrates and substrate deformations (yellow) were determined. northern as well as western analyses for c-Src were performed. These data proof EB2 to be cleaved by γ-secretase and that an EB2 cleavage product is transduced into the nucleus were it functions as transcriptional coactivator for c-Src. Mayer. Furthermore. P. (3). Marambaud. Efthimiopoulos. were used as internal standards Fig. qRT-PCR experiments revealed a strong increase of c-Src mRNA levels in these -/cells compared to untransfected PS1 cells. 034301 119 . Although toxic at high concentrations or enhanced incubation times. (B) -/WT and PS1 cells were transfected with full length EB2GFP and analyzed for nuclear translocation of an EB2GFP fragment. ephrinB1 and ephrinB2. B. classical cell culture employs hard and flat substrates such as polystyrene plates for cell attachment. B. Within micropillar arrays we cultivated rat heart muscle cells (cardiac myocytes). C. Moreover. Cells strongly preferred to span between the elastic micropillars over adhesion to the underlying flat substrate. system was overlayed by a cover slide and PDMS* was crosslinked at 60°C. Heart muscle cells were cultivated within these systems. These highly artificial culture conditions lead to cell adaptation which often severely compromises experimental results.and Nanosystems 4: Biomechanics Marian Smoluchowski Institute of Physics. Reymonta 4. Before use. In addition. Hersch1. Poland In living animals cells are parts of tissues where they experience a soft environment of polymers and neighbouring cells.000 cells were added to a micropillar substrate and analyzed 2 to 3 days after. Sylgard 184 (PDMS*) was carefully mixed in a 20:1 ratio (base:cross-linker) and dispensed onto a microstructured SU-8 master. Image was taken at a height of 20 µm above pillar base. Cell force and micropillar stiffness showed a clear correlation indicating amplitude as decisive feed-back signal for the control of cellular contraction. To this end we derived closed expressions for the stiffness of elastically founded pillars. 1: Myocytes stained for α-actinin (red). In contrast. FIG. Right: Mycocyte grown on flat substrate besides micropillars. Hoffmann1. Note the identical scales. However. Cesa1. Subsequently. Cells were either fluorescently labelled with calcein or fixed for immunofluorescence experiments. Myocytes were isolated from 19-day old Wistar rat fetal pups as described [2]. On flat parts of the substrates we observed prominent stress fibres and focal adhesion sites. N. 120 . R. Krakow. These observations argue for close to nature environmental conditions within elastomeric micropillar arrays. these micropillars could be exploited to determine cell forces. Pillar diameters. Left: Myocyte suspended between micropillars. cells suspended between micropillars exhibited well organized myofibres and costameric adhesions at the locations of Z-bands as found in heart tissue.Heart Muscle Cells Suspended Between Elastic Micropillars A. LSM510). Kirchgeßner1.2. spontaneous myocyte beating lead to micropillar bending that was exploited to determine cell forces. Kajzar1. Merkel1 1 2 Institute of Bio. Note that all sarcomer units are aligned in parallel forming the classical striated muscle morphology whereas this morphology is severely disturbed on flat substrates (right). Side views. Analyses were performed on a confocal microscope (Zeiss. N. Here we developed micropillar arrays from soft rubber as cell culture systems. A comparison of these cells with others that were grown on flat surfaces of identical material revealed much closer to nature cell morphology on micropillars. cover slides with PDMS micropillars on top were peeled off the masters and glued to the bottom of perforated Petri dishes. Furthermore. Jagiellonian University. FIG. 30-059. 30. micropillars were fluorescently labelled with DiD [1]. The next day.M. the architectures of the cytoskeleton and of protein complexes formed for adhesion were strongly dependent on the environment of the cell. 2: Laser scanning microscopy images of pillars (red) with adhered cells (green). 10 µm. This approach revealed that micropillars emerging from identical material are by about 30% softer compared to micropillars with clamped bases. As a further benefit. we determined cell forces from micropillar bending. Sci. Therefore we derived an approximate treatment based on the Boussinesq theory of the deformation of an elastic half space under the influence of mechanical forces.M. 3: Myocytes were grown within labelled micropillar arrays for two days. Freshly isolated myocytes were seeded on these arrays and analyzed.. slope 0.S. Kirchgeßner.g. B. Schwarz. the commonly made assumption overestimates cell forces substantially. Merkel. C. Despite the fact that pillar bending is a classical application of continuum mechanics. Applications in functional drug studies on the single cell level are foreseen. named costamers [3]. 1. Lewis. cf. 78:034301 (2007) [3] Alberts. Full line: contraction forces.H. Cesa. N. Bray and J. classical culture dishes. Molecular Biology of the Cell. Kirchgessner. Fig. broken line: forces in the relaxed state. C: Forces of many cells during contraction (filled boxes) and in the relaxed state (open boxes). In addition. cells grown within micropillar arrays avoided adhesion to the elastic base of the micropillars. In order to characterize the force generation system of myocytes grown between elastic micropillars in more detail. cells were characterized by a strongly reduced number of actin stress fibres. Fig. J. Instrum. these microsystems enable reliable cell force measurements and valuable insight into mechanical regulation of cellular processes.M. B. 3. They are characterized by repetitive units of actin-myosin fibres. Instead. C. no closed expressions for the stiffness of micropillars emerging from a substrate of identical material were available. D. This can be easiest explained by cell contractions being regulated to achieve a specific amplitude. In contrast. Using a confocal microscope pillars were imaged at a height of 15 µm above the base. FIG. B. Myocytes are a highly organized cell type in heart tissues.Freeman & Co Ltd.probe the impact of substrate geometry on these cells.. A. i. A: Pillar positions during cell relaxation (top).6 µm. cf. Mayer. To [1] Kajzar. N. Taken together our data clearly show that close to nature cell culture can be achieved using elastic micropillars. Evaluating cell forces we found a clear cut correlation between micropillar stiffness and cell forces. slope 1. The lines are linear regressions.e. Hoffmann and R. D. neither the high organization of sarcomers nor costameric adhesion structures can be found when myocytes are cultured on flat and stiff substrates as e. Oxford (2006) 121 . W. B: Micropillar displacements along (full lines) and perpendicular (broken lines) to cell orientation. Furthermore. myocytes exclusively span between the pillars for pillar distances of 10 to 30 µm (Fig. 2). called sarcomers. we developed an array of elastomeric micropillars as cellular environment. U. a conclusion which was further supported by the fact that the slopes of the regression lines coincided with the mean cell contractions in relaxed and contracted state. PMID: 17981895 (2007) [2] Cesa.. Hoffmann and R. Biophys. Merkel. Size of myocytes adhered to micropillars decreased compared to those adhered to flat substrate. and contraction (bottom). Rev. These units fill the whole cell with parallel orientation to each other and are connected to the environment by specific adhesion structures.6 µm. An abundance of proteins and signal cascades contribute. the decrease could be attributed partially to introduction of obstacles [2] and partially to viscous effects [3]. For neutravidin the fate of the receptors during membrane adhesion was additionally monitored. well-defined model systems were developed. This effect scaled with the concentration of receptors (see Table 1). R. We study quantitatively the role of the membrane fluidity and the diffusivity of membrane-bound receptors in biomembrane adhesion applying bleaching techniques. Nevertheless. recognition and transport. FIG. Merkel 1 IBN-4: Biomechanics Centre Interdisciplinaire de Nanoscience de Marseille. Membrane adhesion is characterized with respect to the inter-membrane distance by high precision micro-interferometry. We applied Continuous Photobleaching (CP) and Fluorescence Recovery after Photobleaching (FRAP) to study membrane fluidity as a function of specific protein binding to ligands within the membrane and depending on binding of a second membrane. we present a biomimetic model system for cell-cell adhesion mediated by mobile receptor-ligand pairs. prepared by electro-swelling. the viscous protein layer on the distal side of the SLB introduced extra friction. Moreover. right: E-cadherin-E-cadherin. The scope of this project was to elucidate the underlying physical principles. Fenz . which diffused more slowly than single lipids or was completely pinned depending on the receptor concentration. Diffusion thus plays a vital role in many membrane related functions including adhesion. Gibbs free energy of bonding ∆G0 in solution ∼ 35 kB T) binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids reflecting a decrease in the overall membrane fluidity [1]. CINAM/CNRS-UPR3118. traditional Reflection Interference Contrast Microscopy (RICM) theory was extended to account for the reflections from all five relevant interfaces. We used biotinylated lipids to bind neutravidin and NTA lipids to bind the E-cadherin Fc chimera via its histag (for details on the chimera see FIG 3). In recent years. in early stages physical forces dominate as the cell needs some time to initiate an active response. Diffusion of lipids and proteins in the plane of the membrane impacts directly on the local dynamic structure of the membrane. K. A solid supported lipid bilayer (SLB) served as a model for the first membrane. From theoretical considerations. France Cell-cell adhesion is a highly complex process of vital importance for any multicellular organism. To determine the resulting inter-membrane distance with high precision (± 4 nm). To quantitatively investigate the impact of individual parameters on the fluidity and structure of the membrane. 1: We found that in case of strong receptor-ligand interaction (biotin-avidin. 2 . Here. Membrane adhesion was effected either by biotinneutravidin (an avidin analogue) or the extracellular domains of the homophilic cell adhesion molecule E-cadherin (see FIG 1). Sengupta 1 1 2 1.Diffusion and Membrane Adhesion: Avidin and E-Cadherin Case Study S. 122 . were employed to mimic the second membrane. The obstacles were formed by biotinylated lipids pairwise connected to the same neutravidin molecule. Left: biotinneutravidin. Giant unilamellar vesicles. This formed one large object. Sketches of the model systems illustrating the membranes and binding molecules involved. It was prepared by the LangmuirBlodgett Langmuir-Schäfer technique and contained a controlled percentage of ligand lipids. structure and dynamics of the essentially fluid cell membrane and its nanoscale inhomogeneities have attracted much interest. Fenz.2±0. In order to understand the lack of retardation after vesicle binding the inter-membrane distance for both cases was analyzed.6 nm). b) The high precision micro-interferometric measurements of the inter-membrane distance allowed to shed light on the hitherto unclear binding configuration of the cell adhesion molecule E-cadherin. (c) Fluorescence micrograph of accumulated neutravidin in the adhesion disc. At full coverage the receptors were practically immobilized [1]. 1: Specific binding of a GUV membrane led to additional slowing down of tracers (up to 15%) caused by an increase in obstacle density and enhanced friction due to the adhering membrane. ∆G0 ∼ 2 kB T) no significant change in diffusion of tracer lipids was observed upon protein binding and subsequent vesicle binding [1]. the three outmost domains or all domains. hth.2 2. as in the strong binding case.5±0. The scalebar is valid for a-c: 10 µm.8±0.2 5% 2. R. mobile obstacles in the form of coupled NTA lipids. We measured hexp = (54 ± 9) nm strongly favoring the scenario with only the outmost domain involved [1]. At first glance. FIG. Biophysical Journal 52(6). the size of the weak protein-lipid complex was only one third of the strong one. As a result. So far. inter-membrane distance and receptor accumulation should contribute to the understanding of equivalent phenomena in cell-cell adhesion. 123 .2 Diffusion constants [µm2 /s] of the tracer lipid in the SLB before and after binding of protein or vesicle for various concentrations of biotinylated lipids in the SLB.biotin bare +NAV +NAV+GUV 1% 2. Receptors were accumulated in the adhesion zone till full coverage corresponding to 5% biotinylated lipids was achieved (see FIG 2). In summary. Consequently. Evans and E. 3: Three different binding configurations of Ecadherin-E-cadherin are under discussion: overlap of the outmost domain. Nevertheless. 1074-1085 (2009). Langmuir 25(2). (a) RICM micrograph of an adhering vesicle.especially on the number of binding sites and their distance as well as the closeness of the interaction. Sackmann. [3] E. we gained three new insights: a) The impact of protein binding and protein mediated membrane binding on the fluidity of a membrane depends strongly on the geometry of the protein . These results on diffusion.3 = 36 nm. 553561 (1988). it had no measurable effect on the tracer lipid diffusivity. 194. since the Ecad construct is a dimer and its binding should therefore have introduced. flexible membranes bind predominately with their first domain. 2: In case of weak receptor-ligand interaction (Ecadherin-E-cadherin. Error bars represent single standard deviations. F. J. Thus. We measured hexp = (7 ± 1) nm for biotinneutravidin. We conclude that E-cadherin molecules residing on a soft.1±0. The exact E-cadherin binding configuration is still under discussion. c) The adhesion assays in a cell-free model system unequivocally show that accumulation of mobile receptors does not require active interaction of a cell.3±0.1 = 52 nm.2 1.2 1. 989-997 (1987). This height was in very good agreement with the protein and linker dimensions known from crystallography and X-ray reflectivity (hth = 5.2 = 44 nm and hth. (d) Intensity profile along the white line in c. Merkel and K. Fluid. FIG. Mech. [2] M. the adhering membrane generated less friction. TAB. J. three different scenarios were proposed (see FIG 3) corresponding to hth. the lack of retardation after receptor binding is surprising.2 1. [1] S. (b) Reconstructed height [nm] in the adhesion disc. Saxton.7±0. Sengupta. Bleaching experiments on the receptors themselves revealed a fast decrease in receptor mobility with increasing receptor concentration. the inter-membrane distance in the E-cadherinE-cadherin case was significantly larger than in the biotin-avidin case. The initial biotin concentration on the SLB was 2%. M. Since now. so called filopodia. filopodia are thin membrane extensions comprising tight bundles of parallel actin filaments with their proximal end embedded in the lamellipodial actin network. To analyze if adhesion structures can already be found in filopodia the protein localization of focal adhesion site specific proteins 124 . Scale bar=10 μm. Scale bar=10 μm. 1: Correlation between filopodia and focal adhesion sites. These FAs were formed along the axis of filopodia containing all tested adhesion proteins and were just increased in size when reached by the lamellipodium. The given data strongly argued for an important function of filopodia in adhesion site formation of moving cells. wound healing and immune defence. Here we show that nearly all FAs depend on stable filopodia. Many motile cells such as keratinocytes exhibit a highly dynamic formation and disassembly of adhesion sites. in direction of migration (see FIG. Direction of keratinocyte movement=white arrow. keratinocytes were transfected one day before EGF stimulation and subsequently analyzed by confocal microscopy. In contrast. Born. The exact interconnection between focal adhesion (FA) formation and correct guidance is the basis for an efficient functioning of these major biological processes. FIG. We found an essential role for filopodia in the formation of FAs during cell migration. C. Strikingly. While migrating they form a wide lamellipodium with long finger-like structures. Blocking filopodia fully inhibits FA formation. GFP– VASP (E) and GFP–zyxin (F) were analyzed in living cells. talin (B) and vinculin (C). Confocal images in phase contrast (A) and in reflection (B) are shown. Eibl. lamellipodial adhesions were always located in direct extension of filopodia (see FIG. Hoffmann IBN-4: Biomechanics Cell migration is a decisive prerequisite for embryogenesis. Schäfer. The lamellipodium as characteristic feature at the leading edge of motile cells creates the necessary force to pull the front of the cell in direction of migration via actin polymerisation [1] and locates the origins of new focal adhesions [2]. Keratinocytes are epithelial cells derived from human skin which effectively migrate after stimulation. FAs were thought to be built up in the lamellipodium. Borm. In our experiments we focus on the interrelation between filopodial and lamellipodial adhesion structures. E. 1). Note that all proteins can be found in the filopodia extensions. FAs connect the cytoskeleton of the cell to the environmental matrix to provide anchorage and force transmission. 2: Protein content of filopodial extensions. These adhesion sites become prominent at the leading edge of lamellipodia with an elongated shape upon movement. filopodia are the key factors for formation and localization of all FAs and thus for accurate cell movement and direction. Keratinocytes were grown for one day and subsequently motility induced by EGF (epidermal growth factor). Here. B. Möhl. filopodia are described as structures mainly probing the substrate for proper matrix composition but how filopodia integrate the substrate information into lamellipodial processes especially formation of new adhesion sites is still unclear although association of adhesions with filopodia has been described [3]. White arrows indicate fluorescent signals within filopodia in fluorescence and phase contrast. Therefore. Motility induced keratinocytes were fixed and immunofluorescently labeled using antibodies against focal adhesion site specific proteins paxillin (A). Note the localization of focal adhesion sites right behind stably adhered filopodia (black arrows in A and B). Functionally. 1 A).New Role for Filopodia in Adhesion Formation During Cell Migration C. FIG. Localization of GFP–tensin (D). Lamellipodial adhesions were always found right behind stable filopodia whereas cell areas with filopodia unable to attach were characterize by the absence of lamellipodial adhesions. S. B. 180 (2008) 1233–1244 C. Cells transfected with GFP–vinculin identified a full correlation between the neomycin induced migratory block and inhibited focal adhesion formation. Mean value and standard deviation for every time point from normalized curves are given (blue. For this reason we blocked filopodia formation by neomycin supplementation. M. Danuser. the vasodilator-stimulated phosphoprotein (VASP) and vinculin was tested by immunofluorescence as well as in living cells using green fluorescent protein (GFP)-fusion constructs (see FIG. These data clearly show that each of these proteins is localized in filopodia most likely along their complete lengths with signal intensities highly above cytoplasmic background. (B) GFP–vinculin transfected cells were treated with neomycin. Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella. [1] A. Same position is also indicated in phase contrast in the first image of the series. During this enlargement process the spatial orientation of the focal adhesion did not change. Merkel. Diez. G. neomycin untreated cells (red. C. Therefore we analyzed adhesion formation in filopodia using live cell imaging (see FIG. Küpper. Time points are given in seconds. Goldmann. focal adhesions at the leading edge were analyzed by FRAP. Cell Sci. Their localization labeled parts of the filopodia or covered their whole lengths but was always elongated. neomycin untreated keratinocytes while nascent adhesions of migrating cells contained a much higher fraction of exchanging vinculin (see FIG. Kirchgeßner. B. FRAP (Fluorescence Recovery After Photobleaching) analyses on these cells revealed a full maturation state of focal adhesions formed before neomycin treatment but still located at the leading edge of polarized cells (see FIG. all tested adhesion proteins were present along stably adhered filopodia. We clearly showed that adhesion structures can already be found in filopodia. 3).001). For better visualization data were overlaid with experiments on focal adhesions at the leading edge of migrating. Taken all these data together we present new insights in formation and function of filopodia in migrating keratinocytes. 2). talin. Here. Two distinct actin networks drive the protrusion of migrating cells. G. Zaidel-Bar. Machacek. These results were highly comparable to those found in [4] for focal adhesions of sessile. Migration speeds are significantly different (pvalue<0. J. tensin. n=14. Ponti. 2 F). Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. Vinculin exchange dynamics regulates adhesion site maturation and adhesion strength. Geiger. A single exponential model was fitted to the data describing a simple binding-unbinding kinetics. red curve). M. Furthermore. Z. Waterman Storer. 4 A). Möhl. Afterwards. unpublished. Since our data on moving keratinocytes clearly showed a dependency of focal adhesion sites on filopodial adhesions. These data suggest that stable filopodia form small but fully assembled focal complex like structures termed here filopodial focal complexes (FX). FIG. W. FIG. Nemethova. Science 305 (2004) 1782–1786. S. J. 4 B. Hoffmann. C. 4 B). [4]). Stably adhered filopodia were analyzed over time in phase contrast (top) and fluorescence (bottom). n=13). the size of the adhesion structure increased along the former orientation of filopodia. Note the strong increase in fluorescence as soon as the lamellipodium reaches the filopodial adhesion.M. Keratinocytes were transfected with tensin as GFPfusion protein and motility stimulated after 24 h of growth. It becomes clear that filopodia play a fundamental role during adhesion site formation and thus determine the localization and shape of almost every adhesion site. we analyzed whether focal adhesions would be formed in the absence of filopodia. R.H. Kam. C. 4: Blocking filopodia inhibits motility and adhesion formation: (A) Migration speeds of EGF stimulated keratinocytes with (n=74) and without (n=62) neomycin treatment were analyzed and are indicated as boxplots. Ballestrem. [2] [3] [4] 125 . Schäfer.V. As soon as the filopodial adhesion proteins were reached by the lamellipodium. Small. 116 (2003) 4605–4613. These data illuminate that form and localization of focal adhesions is determined by filopodial adhesions. Scale bar=3 μm. N. a marker protein for mature focal adhesions (see FIG. Auinger. In the presence of neomycin all cells (n=338) were significantly reduced in migration (see FIG. J. Gupton. K. B.like paxillin. To our surprise the same localization was also found for zyxin. Cell Biol. R. 3: Growth of filopodial FXs to focal adhesions. S. White arrows indicate filopodial FXs at time point 0 changing to focal adhesions over time.L. C. R. During their maturation they increase in size remaining relatively stable in respect to the substrate. We investigated the phosphorylation and exchange dynamics of the FA protein vinculin in epithelial cells from human skin (keratinocytes) migrating on a flat surface. 1). Kirchgeßner1. we photobleached vinculin at these sites and measured the fluorescence recovery over time. Therefore. vinculin connects other FA proteins with actin filaments and is thought to stabilize the adhesion complex [2]. To measure the vinculin exchange in single FAs. K. As an adaptor protein. A: Pseudocolor image of the ratio between phosphorylated vinculin and total vinculin within FAs of a migrating cell. If there was no protein exchange. Möhl1. Hence. W. with this method called Fluorescence Recovery after Photobleaching (FRAP) the amount of the stably incorporated against the constantly exchanging vinculin could be determined in single FAs (see FIG. Many proteins incorporated in FAs are constantly exchanging within a timescale of seconds. Asterisks indicate significant differences of vinculin phosphorylation compared to FAs of stationary cells. since their movement is based on a constant adhesion turnover with formation of new adhesions at the front and the release of old adhesions at the cell’s rear. if all bleached proteins were replaced. Migrating cells are excellent model systems to study the controlled assembly and disassembly of FAs. FAs undergo a regulated development from a nascent to a mature state by changing size. that the exchange of the FA-protein vinculin decreases with proceeding age of FAs and is regulated by phosphorylation at a single tyrosine residue of vinculin. Thus. Based on these findings we consider the phosphorylation-regulated incorporation of vinculin as a mechanism to modulate adhesion strength. the fluorescence would recover completely. shape and protein composition. Hoffmann1 1 2 IBN-4: Biomechanics Center for Medical Physics and Biology. Nascent FAs are formed at the front of the migrating cell. Diez2. migrating keratinocytes were transfected with a gene for fluorescently labelled vinculin making it possible to visualize this protein in vivo by light microscopy. Quantitative immunostaining studies revealed a high vinculin phosphorylation in the nascent FAs at the cell front. the coordinated formation and release of FAs determines the direction of migration and is thought to play a key role in directed cell movement like chemotaxis [1]. the fluorescence wouldn’t recover at all. 2 A). The mechanical connection between the actin cytoskeleton and the ECM is provided by protein clusters at the cell membrane building distinct adhesion spots which are denoted as focal adhesion sites (FAs). N. Data were collected from 38 cells. The cell shape is marked by a white line and the big arrow points to the direction of migration. While the cell-body moves forward they come closer to the trailing edge of the cell where they finally dissolve.H. G. Friedrich-Alexander-University of Erlangen-Nuernberg maturation state. Error bars indicate standard deviation. We therefore propose a new role of vinculin as a tuneable factor for the mechanical stability of FAs. 1: Phosphorylation of vinculin in focal adhesions (FAs) of migrating skin cells. This stabilization of vinculin within the FA goes along with an enhanced force transduction ability of the cell. During their lifetime. Since phosphorylation events are known as a mechanism to regulate the binding ability of certain proteins. With increasing FIG. Schäfer1. B: Amount of vinculin phosphorylation in FAs in respect to their distance from the leading edge. Here we show. The morphogenesis and architecture of multicellular organisms is determined by the coordinated adhesion of single cells to each other and to the extracellular matrix (ECM). Küpper1.The Mechanism of Adhesion Strength Adaptation in Living Cells C. 126 . Nascent FAs at the cell front (arrowheads) are higher phosphorylated than mature FAs located behind. Merkel1. In contrast. Goldmann2. the phosphorylation of FAs was significantly lowered (see FIG. B. we explored the exchange kinetics of vinculin in respect to the FA maturation state. 2 B and C). Nascent FAs at the front produce weak rearwards directed forces. A: FRAP experiment at a mature FA of a cell transfected with fluorescently labelled vinculin. Hoffmann B. where phosphorylation at a specific tyrosine residue (TYR1065) was inhibited. Scale bar: 5 µm. Scale bar: 10 µm. To verify this theory. Schwarz US. the regulation of vinculin binding could be a mechanism to adapt the adhesion strength of individual FAs. FAs appear as bright spots. n=14). Therefore.3: Force application during cell locomotion: Fluorescent image of vinculin in a migrating cell with FAs appearing as bright spots. Taken together. The white arrow points to the direction of migration. nascent FAs with phosphorylation-inhibited vinculin (grey. 3). Thus. Merkel R.FIG. these experiments confirm the direct interplay between the amount of stablely incorporated vinculin into the FA and its ability to transduce force. traction forces could be calculated from these deformations. C: Saturation values of the mean recovery curves shown in B. The exchange of the inhibited vinculin mutant was in nascent FAs significantly lowered compared to the wild type vinculin confirming the direct interplay between the phosphorylation of vinculin and its binding kinetics (see FIG. This value indicates the exchanging fraction of vinculin. mature FAs at the rear (green. n=11). Since the elastic properties of the silicone substrate and the location of FAs as sites of force transduction were known. cells were transfected with a slightly altered vinculin gene. B: Mean fluorescence recovery of vinculin over time from several FRAP experiments: nascent FAs at the cell front (red. [1] Webb DJ. n=13). the fluorescence recovers over time.2: Vinculin exchange dynamics examined by Fluorescence Recovery after Photobleaching (FRAP). FAs from sessile cells (blue. Horwitz AF. FIG. whereas the vinculin incorporation could be modified by phosphorylation. n=14). the correlation between FA maturation state and force transduction was examined by traction force microscopy of cells migrating on elastic silicone substrates [3]. we suggest vinculin to be an important stabilization factor for FAs which is directly regulated by phosphorylation through intracellular signalling pathways. Just like the amount of phosphorylation. Rev Sci Instrum 78(3):034301 (2007) 127 . Red arrows mark the force vector for each FA. Eigenvectors of the generalized first moment tensor are indicated by green arrows Mature FAs at the rear produce strong inwards directed forces. Kirchgessner N. Trends Cell Biol 16(9):453-60 (2006) [3] Cesa CM. These experiments revealed that major traction forces were transmitted at the cell’s rear where typically mature FAs are located (see FIG. the fraction of exchanging vinculin decreased with the FA maturation level indicating that vinculin exchange is regulated by phosphorylation. Since vinculin is constantly exchanging within the FA. tractions of the migrating cell produced local substrate deformations which could be quantified by tracking the displacement of fluorescent beads embedded in the substrate. Nat Cell Biol 4(4):E97-100 (2002) [2] Ziegler WH. Critchley DR. Liddington RC. Parsons JT. The bleach-field is marked by a red circle. As Vinculin is essential for mechano-coupling. Mayer D. Asterisks designate a statistically significant difference from frontal (red) or rear (green) adhesions. In these experiments. Bleaching occurs at 0s. 128 . Education & Dissemination International Helmholtz Research School International Soft Matter Conference 2007 IFF Spring School 2008 Jülich Soft Matter Days 129 . independent talks on different topics: Monte Carlo and Molecular Dynamics Simulations. the group ’Molec- 130 . Vliegenthart. their non-equilibrium behaviour and their response to external fields. electrophysiology. there is an urgent need for a more quantitative. Colloids. The fellow PhD students will be based in one of the groups that are part of the IHRS. life science research has undergone a fundamental transition. the students learned about the important tool of ’Computer Simulations in Physics and Biology’ by a two-semester advanced-seminar course that covered various. Merkel. S. run in cooperation with the universities in Cologne and Düsseldorf and caesar Bonn. Kaupp. Mesoscopic Hydrodynamics. theory-oriented approach. and Membrane Proteins. Winkler. A. Egelhaaf in Düsseldorf. Willbold. Dr. multidisciplinary research centre. A particular focus is laid upon unraveling the physics of biologically relevant systems. It also offers a comprehensive framework of experimental and theoretical techniques that will enable PhD students to gain a deeper understanding of the structure. Polyelectrolytes. Kaupp in Bonn. Students benefit not only from lectures. G. R. Enderlein. It has become evident that even the simplest molecular machines display an astounding complexity. Furthermore. two-semester introductory lecture courses. Most of the speakers were from Forschungszentrum Jülich and daily work with the methods and systems they presented: G. made great progress in understanding the structure of complex multi-component macromolecular systems. fluorescence spectroscopy. U. dynamics. and biology in research and education. ProteinLigand Binding. and lab courses given by experts in the field. leaving alone networks of genes and proteins in a living cell. and Prof. Schurr. Lercher. Gompper and ’Molecules of Life Introduction to the Chemistry and Biology of Cells’ with various lecturers from within the IHRS BioSoft (Prof. Both courses equip the students for their research projects with important basic knowledge: from a physics point of view. A. Seidel in Düsseldorf. Ripoll. and the physical chemistry group headed by Prof. Evolution of Bacterial Genomic Networks. but also from courses in transferable skills. G. and function of complex systems. there is an urgent need for an interdisciplinary graduate education. Büldt. Baumgärtner. chemistry. M. Seidel. In recent years. the group on ’Molecular Physical Chemistry’ headed by Prof. Prof. Heise. but also participate in interdisciplinary courses. The lectures of the school usually attract a number of extra participants that choose the topics selectively according to their needs. Soft matter research has. the ICG-3 (Phytosphere) headed by Prof. The biological lectures covered systems from amino acids to the structure and dynamics of entire cells as well as methods such as X-ray crystallography. Proteins. Solid State NMR. Thus. ’Introduction to Statistical Physics’ taught by Prof. ular Sensory Systems’ headed by Prof. Dhont and Prof. Strey in Cologne participate in the IHRS BioSoft. C. in parallel. Offenhäusser) were offered to the students. In addition to groups within the PoF-BioSoft program. Other students are welcome to join most of these courses as long as there are free places. In 2007 and 2008. Richter. Prof. M.International Helmholtz Research School of Biophysics and Soft Matter The International Helmholtz Research School of Biophysics and Soft Matter (IHRS BioSoft) provides intensive training in biophysics and soft matter. Naegele. they experience the environment provided by a large. M. entropy and statistical physics have a large influence on the behaviour of the systems that are usually mesoscopic. Prof. seminars. M. B. and optical microscopy. U. Its ultimate goal is to advance the integration and exchange between physics. H. Prof. Schurr in Jülich. Protein Structure Prediction. In 2007. and funded by the Helmholtz Association. Prof. Thus. Zacharias The IHRS BioSoft is located at Forschungszentrum Jülich. Stoldt. The research school accepts fellows for three-year PhD projects and is open to highly qualified and motivated applicants from all countries. Prof. also the group ’Physics of Soft Matter’ headed by Prof. chemists. In the last semester. Aho. Frank. Molecular Sensory Systems. Poire. students were offered the onesemester introductory lecture course on ’Cell Biology’ by Prof. The PhD students regularly present their research in the Student’s Seminar that is chaired by two IHRS BioSoft faculty members. Theory of Soft Matter and Biophysics) • Photo-control of cell networks for extracellular recording systems (V. first students who have started their PhD projects within the IHRS BioSoft finish their thesis. Brückel. and the advanced lecture courses on ’Complex Fluids’ by Prof. Fischer (NYMU. Biomechanics) • Swarm behaviour of self-propelled particles (Y. Molecular Physical Chemistry. Physical Chemistry. 131 . Cologne) • Squeezing actin: a TIRF microscopy study (A. Bonn) • Morphologic and physiologic aspects of synaptic transmission in rat barrel cortex (G. Seidel in Düsseldorf was open for IHRS fellows. Strey (Cologne) and ’Rheology’ by Prof. The course provides an extensive training by theoretical lectures and practical exercises. In a course on ’Optical Spectroscopy’. Sauer (Bielefeld). Brodeck. Düsseldorf) • How is shoot growth affected by low root temperature? (R. also the research topics of the PhD projects cover a wide range within biophysics and soft matter. J. Haack. Monzel. Molecular Sensory Systems. Brüggemann. and biologists can benefit. such as fluorescence techniques. Schuetz (Linz). • NMR as a tool to study protein structures (M. Bonn) • Nanostructured gold electrodes for the functional coupling with neuronal cells (D. The laboratory course ’Recording of Cell Activity’ — for example on Ca2+ imaging in living cells — was offered jointly by several institutes within the IHRS. Tsigkri. presentation techniques up to writing of applications towards the end of the PhD project. Humplickova (Prague). Grabowski. Structural Biochemistry) • Non-genomic action of progesterone in human sperm (N. Physical Chemistry. the speakers usually receive also comments regarding the style of the presentation and whether it was suitable for the different parts of the audience. Enderlein (Tübingen). and J. J. Cellular Neurobiology) Fellow PhD students already participated in two of the three seminars in transferable skills by Imperial College London that are organized by the Helmholtz Association. As the research in the participating groups. J. Zorn) is open for the participation of IHRS students. B. T. Belkoura and M. a two-week course ’Fluorescence Spectroscopy’ by Prof. Müller and Prof. Savic. Goodwin. every talk is followed by a long discussion. Vermant (Leuven). Richter. Bioelectronics) Complementary laboratory courses provide the students with practical experience and strengthen the interdisciplinary approach. Gensch. Granzin. a one-week course ’Cryo Transmission Electron Microscopy’ was organized exclusively for IHRS students by L. Theory of Soft Matter and Biophysics) • Molecular Dynamics simulations of polyethylene oxide (PEO) and PEO/PMMA blends (M. The seminars shall cover various aspects ranging from group work in the beginning of the thesis. Hanes. M. Very recently.(IU Bremen). Soft Condensed Matter) • Microemulsions as delivery systems (Sabine Schetzberg. and W. Topics of talks that were given include: • Holographically induced nucleation (R. and Raman spectroscopy of biomolecules. Bonn) • Self-assembly in a binary H2O-C12E4 system (I. Yang. König in Jülich. Bioelectronics) • Microinterferometry: a tool to study membrane fluctuations (C. single-molecule spectroscopy. Currently. Hofkens (Leuven). Therefore a talk in the Student’s Seminar is very challenging. and R. Düsseldorf) • Regulation of HCN channels by phosphorylation (F. Schwarten. because it needs to be prepared such that physicists. Apart from questions and feedback about the research. G. Molecular Sensory Systems. Maybeck. Taiwan). Physics of Soft Matter. Heger. Baciu in Cologne and a one-day course on ’NMR Spectroscopy’ was offered by B. the two-week ’Neutron Scattering’ course (organized by T. Every year. Phytosphere) • HCN channels in the main olfactory bulb (A. Baumann that included lab demonstrations. reaction-induced infrared difference spectroscopy. G. J. D. Heberle (Bielefeld) taught several optical techniques. Cologne) • Polyelectrolyte electrophoresis (S. Winkhaus. Neutron Scattering) • Combined single-molecule force and fluorescence spectroscopy (S. or the modification of the properties of polymers melts by addition of colloidal particles to form nano-composites. phonons and melting of colloidal crystals. biomaterials and their composites [2. The need for a unified view of Soft Matter systems is threefold. For even larger aspect ratios. rods typically become more flexible. 1]. Instead. we arrive at the classical. roughly speaking.K. the application of physical concepts and ideas to biological systems has become one of the most intense activities in soft condensed matter in recent years. becomes larger. Leibler (ESPCI. colloids are hard. D. for which DNA is an example of enormous importance. 2007. 1. we hope that the list of the plenary speakers and the titles of their talks will give a feeling about the exciting atmosphere and the intensity of the discussions during the conference: • M. It brought together about 600 scientists from 35 countries to discuss all aspects of soft matter science. Richter 3 1 2 3 IFF-2: Theoretical Soft-Matter and Biophysics IFF-7: Soft Condensed Matter IFF-5: Neutron Scattering The International Soft Matter Conference took place in Aachen on October 1-4. mixtures of several components of colloidal. colloids. 2). polymers and surfactants are by far not as distinct materials as previously assumed. As the aspect ratio. which fills the triangle of materials illustrated in Fig. First. Cates (University of Edinburgh. 85 contributed talks. USA) Physical aspects of viral infectivity. 1. 42 invited talks. and surfactants. because they open up the possibility to tune and control material properties. Germany) Elasticity. amphiphilicity as abscissa and elongation or flexibility as ordinate. • W. there is essentially a continuum of molecules and systems. • L. in the limit of very small persistence lengths. Traditionally. Let us illustrate this by following the left-hand side of the triangle from colloids to flexible polymers. membranes. Dhont 2 . • G. etc. Gompper 1 . Los Angeles. Aachen 2007 G. and about 400 posters. With more than 600 participants from about 35 countries (see Fig.International Soft Matter Conference. An example is the fd-virus shown in Fig. 1: The components of modern Soft Matter systems can be arranged in a triangle. With the large number of conferences organized every year on topics like polymer chemistry and polymer physics. which shown that there is a continuum of molecules and materials which fills the space between spherical colloids. flexible polymers. surfactants in solutions. the tuning of membrane properties by anchored polymers and amphiphilic block copolymers. it has been recognized over the last decades that colloids. Indeed. surfactants. Well-known examples are the depletion interaction between colloidal particles induced by polymers in solution. the amount of information provided during the conference was much too large to be summarized in a few lines here. UK) Lattice Boltzmann simulations of nonequilibrium complex fluids. there are also rod-like colloids. flexible synthetic polymers. Maret (Universität Konstanz. Paris. the length exceeds the persistence length. Indeed. the aim of an International Soft Matter Conference clearly had to be to bring together a balanced mixture of scientists working on all kinds of soft materials — such as polymers. J. polymeric or amphiphilic character are becoming increasingly important.. Third. 8 plenary talks.G. Finally. biological and biomimetic systems share many macromolecules and properties with Soft Matter sys- FIG. Second. spherical particles. tems. France) Supramolecular plastics and rubbers. the ratio of rod length and rod diameter. 132 . However. the intriguing mesophases in mixtures of spherical and rod-like colloids. this is the regime of semi-flexible polymers. The two main axes of this triangle are. Gelbart (University of California. colloid chemistry and colloid physics. • D.K. Weitz (Harvard University. drops and wetting: Structuring new soft materials with microfluidics. 133 . J. Roux (Saint-Gobain. Tanaka (University of Tokyo. This issue provides a good overview about the current status and topics of Soft Matter research. USA) Dripping. [3] Special Issue Eur. Japan) Mechanical instability in phase separation. 1 (2008). Courbevoie. Nelson (Harvard University. All invited speakers were invited to submit an original paper related to the subject of their presentation for a special issue of the European Physical Journal E [3]. Eur. J.FIG. [1] European Network of Excellence Newsletter No. Phys. [2] G. 2: Number of participants from countries with the largest participation. Participants were therefore asked for proposals. The conference announcement is shown in Fig. 3. • H. A public vote showed the largest support for the proposal from Granada (Spain). December 2007. jetting. 3: Announcement of the International Soft Matter Conference 2007. These proposals were presented in a plenary session. USA) Neutral mutations and gene surfing in microorganisms. All contributions were fully reviewed. It will therefore the location of the International Soft Matter Conference 2010. • D. • D.G. and D.5. J. Gompper. the program committee came to the conclusion that it would be a good idea to organize international soft matter conference every three years in the future. fracture. Richter. FIG. Phys. Dhont. E 26. E 26 (2008). We are looking forward to this meeting of the Soft Matter community. Due to the large interest and overwhelming participation in the conference. France) Glass technologies: colloids and active surfaces. “SoftComp". and cavitation. and a high sensitivity to external fields. soft nanoscale materials. environmental. Germany. self-organized and biomimetic systems with novel or unusual properties will broaden the spectrum of applications. Structural and novel functional properties of soft and biological materials need to be studied invoking self-organization and hierarchical structure formation. Soft matter is ubiquitous in a vast range of technological applications and is of fundamental relevance in such diverse fields as chemical. The unusual dynamics of complex fluids requires special approaches to gain insight into diffusion transport properties. entropic particle interactions and fluid-like aspects of biological materials such as vesicles and cells.g. and food industry as well as life sciences. Leading scientists from research and industry gave 220 students and young scientists from 25 countries and five continents a comprehensive overview of the interdisciplinary research field "Soft Matter" at the interface between physics. The synthesis of complex materials. chemistry. in life sciences and material processing. addressed advanced experimental techniques and 134 .to millimetres and nanoseconds to days. and external fields.39th IFF Spring School: Soft Matter – From Synthetic to Biological Materials The 39th international IFF Spring School took place from 3 March until 14 March at the Forschungszentrum Jülich. rheology and mesoscopic flow behavior. Soft and biological materials share fundamental structural and dynamical features including a rich variety of morphologies and non-equilibrium phenomena. with spatio-temporal correlations that can span a huge range from nano. an unusual frictiondominated flow dynamics.. • • • The IFF Spring School 2008 at the Forschungszentrum Jülich. e. self-organisation. The exploration of advanced theoretical and computer simulation methods that span the large range of time and length scales and allow to cope with an increasing complexity of molecular constituents. The key requirements for the advancement in the field of these highly complex soft materials are: • The development of novel experimental techniques to study properties of individual components in processes and the cooperative behavior of many interacting constituents. which are influenced by a delicate interplay of hydrodynamic interactions. biology and the life sciences. soft matter science has been largely extended in its scope from more traditional areas such as colloids and polymers to the study of biological systems. and the development of novel composites and microfluidic devices. These properties emerge from the cooperative interplay of many degrees of freedom. thermal fluctuations. Existing methods need to be extended and new approaches are required to describe systems far from equilibrium. Over the past years. Introductory lectures provided the basis of important experimental and theoretical tools. as well as the opportunity to participate in practical courses and visits to the participating institutes at the Forschungszentrum Jülich. More advanced lectures explained practical aspects of various methods and lead the participants from basic methods to the frontiers of current research.applications. radio and television. The local media coverage included newspapers. The lectures covered the following topics: • • • • • • Scattering Techniques Single Molecule Techniques Equilibrium.and Non-equilibrium Statistical Physics Microfluidics Computer Simulations Synthesis • • • • • • • Self-Organisation Flow Properties and Rheology Biomechanics Macromolecules and Colloids Membranes and Interfaces Biomimetic Systems Glasses and Gels The school offered about 50 hours of lectures plus discussions. 135 . and theoretical and computer simulation methods on an undergraduate and graduate student level. Utrecht) and dynamics of charged colloids (G. The experimental and theoretical investigations as well as the understanding of the properties of these materials poses enormous challenges due to their high complexity. In the session on physics of the cell. 1: Announcement of the Jülich Soft Matter Days 2008. Germany.M. München). Kegel. Leeds).Jülich Soft Matter Days 2008 J. Gompper 2 . While many of these systems have already been investigated for a long period of time. In the polymer session. Stuttgart). Kyoto). The topics that have been discussed 2008 in the colloid session range from Archimedian tilings on quasicrystalline surfaces (C. We hope that this conference provided a forum to share and discuss the latest advances for all researchers in this field.J. to buckling of microcapsules (A. Rubinstein.K. synthesis of nanoparticle-microgel composites (L. The number of participants is limited to about 220. Schwarz. Liz-Marzan.P. G. fiber organization in living cells (F. Yoshikawa. amplification of tension in branched polymers (M. in recent years. cell shapes and forces on patterned substrates (U. and fd-viruses are in between rod-like colloids and stiff polymers. Imhof. systems with various types of complex particles have been studied. which ensures an informal atmosphere and avoids parallel sessions. polymer-solutions. 136 . Jülich). -mixtures and -melts. and the rheology of branched entangled polymer melts (D. Richter 3 1 2 3 IFF-7: Soft Condensed Matter IFF-2: Theoretical Soft-Matter and Biophysics IFF-5: Neutron Scattering The Jülich Soft Matter Days is a yearly conference. Nédélec. Vigo).G. Heidelberg). In addition. Russell. Bechinger. binary and ternary amphiphilic systems (microemulsions). colloids at interfaces (W. Dhont 1 . only recently their common features and mixtures have come into focus. Chapel Hill). highlights include talks about capillary wrinkling of floating polymer films (T. held at the Gustav-Stresemann-Institut in Bonn. the main topics were the role of DNA conformations in gene control (R. Janus colloids share features of colloids and amphiphiles. FIG. membranes. D. Amherst). An important part of the conference are two posters sessions with more than 120 contributions. For example. where the particles exhibit properties that are both of colloidal. Metzler. Karlsruhe). the large number of cooperative degrees of freedom. Read. These highly complex systems are characterized by structural units with typical length scales ranging from nanometers to micrometers. Nägele. time and energy scales. and the large range of relevant length. block copolymers. vesicles and biological macromolecules. The systems of interest include colloidal dispersions. The conference attracts scientists from all continents and has a pronounced international character. Likos. polymeric and possibly amphiphilic character.K. Utrecht). multiple glassy states in star-polymer mixtures (Ch. Düsseldorf). the physical and biological significance of transition in DNA (K. The aim of these meetings is to bring together scientists from all soft matter disciplines and from biophysics.N. Kneller. Nakano. 137 . Lund. Finally. the session on hydrodynamics included talks on ordering of colloids by flow and sedimentation (M. and cytoskeleton dynamics of liposomes (C. 2: Discussions during one of the two the poster session. Paris). Rehovot). The posters were grouped according to the same topics as the oral contributions. Bar-Ziv. rheology of microphaseseparated diblock copolymers (T. San Sebastian) and lipid vesicles (M. The main interests in the session on proteins were on neutron scattering to obtain information on structure (D. Ann Arbor). micro-fluidic crystals (R. FIG. Solomon.J. Kyoto). Barrat. Some of the topics of interest in the session on self-assembly were the kinetics of block-copolymer micelles (R.2. microscopic swimmers at surfaces (J. Kyoto). Elgeti. and heat and mass transport at interfaces (J.fz-juelich.de/iff/jsmd2008. Svergun. 3: The participants meet in the central court of the Gustav-Stresemann-Institut in Bonn.An important part of conference are the poster sessions. The more than 120 poster contributions were split into two poster sessions. Hamburg) and inter-domain dynamics (R. which gave all participants the opportunity for lively discussions at the posters. please visit the conference webpage http://www. and protein aggregation in chinese century eggs (E. Ohta. Jülich). Sykes. Lyon). and bacterial swarming (J. We hope that the Jülich Soft Matter Days will continue to be successful as an inter-disciplinary meeting on soft matter and biophysics. Biehl. Leuven). NMR experiments and simulations on self-similar dynamics (G. Cambridge). an impression of which is given in Fig. For more information about the 2008 conference and the upcoming 2009 meeting.I.-L. Jülich). Orléans). Vermant. FIG. Eiser. 138 . Publications 2007 2008 139 . R. 587 .Mathematical and General. Bringer. D. E.*. 198101-1 .12937 Eisenriegler. 045103 Gwan. Baumgaertner. Engineering.. Phase transition in the two-component symmetric exclusion process with open boundaries Journal of Statistical Mechanics: Theory and Experiment. V. M. R.606 Gwan. Schütz. Gompper. 76 (2007). P07020 Harris. Baumgaertner.. 78 (2007).. and Biology. G. Gompper. S. 046705 Grosskinsky. Cooperative Transport in a Potassium Ion Channel Journal of Chemical Physics. Ion Transport in a Nanochannel Journal of Computational and Theoretical Nanoscience : for all Theoretical and Computational Aspects in Science. H. 75 (2007)... 034904 Götze. 79 (2007). 8 (2007).11243 Brzank. G. Polymer depletion profiles around nonspherical colloidal particles Journal of Chemical Physics. 98 (2007). M. G. G. Phase transition in a cellular automaton model of a highway on-ramp Journal of Physics A . J. J. Transport coefficients of dissipative particle dynamics with finite time step Europhysics Letters. Effect of a Low-Dielectric Interior on DNA Electrostatic Response to Twisting and Bending Journal of Physical Chemistry B. 11221 .. F. 127 (2007). Schütz.Institute of Solid State Research Theoretical Soft Matter and Biophysics (IFF-2) 2007 Belitsky. Schütz. A.*.56 Harris. J. A.* Scaling approach to related disordered stochastic and free-fermion models Physical Review E.. F. Schütz.. (2007). R. Kikuchi. 10005 140 . 36002 Noguchi. 031104 Kohyama. M. Fluctuation theorems for stochastic dynamics Journal of Statistical Mechanics : Theory and Experiment. Relevance of angular momentum conservation in mesoscale hydrodynamics simulations Physical Review E.. H. 40 (2007). Gompper. G. Willmann. 50 . 128103 Noguchi. Noguchi. A. G. J. B.. Rigorous results on spontaneous symmetry breaking in a one-dimensional driven particle system Journal of Statistical Physics. M. N. 127 (2007). G. G. Stinchcombe. Swinging and Tumbling of Fluid Vesicles in Shear Flow Physical Review Letters. Maric. 12933 .. A. Particle-based mesoscale hydrodynamic techniques Europhysics Letters. Gompper. T. 128 (2007). 4 (2007). H. R. G. Defect Scars on Flexible Surfaces with Crystalline Order Physical Review Letters. H. Gompper. P08028 Cherstvy. I. G.. A. N. 111 (2007). 98 (2007)....198101-4 Noguchi. 23 (2007). Schütz.J. G. 26 (2008). G.. 1 . G. T. G. Protein ..Ripoll. M. M. G.4750 Cherstvy.430 Winkler. Free energy and extension of a semiflexible polymer in cylindrical confining geometries Physical Review E. M. Cherstvy. Barma. Richter.. R. 112 (2008). S. 349 . R. 1852 . G.. S. and Biology. 76 (2007). G. 111 (2007). R.*. Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy Journal of Chemical Physics.*. 309 .8493 Yang. Lamura. Mesoscale simulations of polymer dynamics in microchannel flows Europhysics Letters. 77 . M. 25 (2008).321 Frank.. 77 (2008). 011804 2008 Baumgaertner.061124-8 Cherstvy. 054904 Winkler. A. Hydrodynamic screening of star polymers in shear flow European Physical Journal E. G. A. 112 (2008). M. 8486 . A. DNA Cholesteric Phases: The Role of DNA Molecular Chirality and DNA-DNA Electrostatic Interactions Journal of Physical Chemistry B. Winkler. Concepts in Bionanomachines: Translocators Journal of Computational and Theoretical Nanoscience : for all Theoretical and Computational Aspects in Science. Discontinuous Condensation Transition and Nonequivalence of Ensembles in a Zero-Range Process Journal of Statistical Physics..1890 Cannavacciuolo. Gompper. G.. G. Winkler. Gompper. A. 132 (2008). G. G.2 Großkinsky.12595 Finken. K. R. 4741 . Y. 127 (2007).. 5 (2008) 9. Adsorption of Weakly Charged Polyelectrolytes onto Oppositely Charged Spherical Colloids Journal of Physical Chemistry B. Polyelectrolyte electrophoresis: Field effects and hydrodynamic interactions Europhysics Letters.. G. R. W.. 061124-1 .. Shock probes in a one dimensional Katz-Lebowitz-Spohn Model Physical Review E. U. G. G. D. Chleboun. 38004 Gompper. Dhont.. Instability of condensation in the zero-range process with random interaction Physical Review E. 030101 141 ..*. 83 (2008). Winkler. Harris. Gompper. J. Two-dimensional fluctuating vesicles in linear shear flow European Physical Journal E. 83 (2008).. R. Engineering. L. Gompper.108 Grosskinsky. R. 34007-1 . S. Schütz.* Hydrodynamics of the Zero-range Process in the Condensation Regime Journal of Statistical Physics. 78 (2008) 3. Seifert. G. Burkhardt. A. 127 (2007) 2.34007-6 Chatterjee. 12585 . S. Editorial : A unified view of soft matter systems? European Physical Journal E.DNA interactions: Reaching and Recognizing the Targets Journal of Physical Chemistry B..354 Schütz. 419 . P. Winkler... Mesoscale hydrodynamics simulations of attractive rod-like colloids in shear flow Journal of Physics: Condensed Matter.. Exact solution of a stochastic susceptible-infectious-recovered model Physical Review E.. G. C. 128 (2008). E. P. 034502 Noguchi. G. 78 (2008) 1. H. G. Holmqvist. Heresanu.. Gompper. Trimper. Rouziere. Gompper.. M. 16 (2008). R. P. Winkler.O.168302-4 Ripoll. M. V.300 Vliegenthart. 191 – 199 Huang..144902-12 Verberck. Y. 14 (2007). M.*. 78 (2008). Asymmetric simple exclusion process with periodic boundary driving Physical Review E. J.. G. Schütz. S.Harish.. 144902-1 . M. G. Gompper. 101 (2008).. 111 . J. G. Dhont.. K. G. K... Pekker. 061132 Tao. G... Götze. Attractive Colloidal Rods in Shear Flow Physical Review Letters. 061903 142 . Transport coefficients of off-lattice mesoscale-hydrodynamics simulation techniques Physical Review E. Multiparticle collision dynamics modeling of viscoelastic fluids Journal of Chemical Physics. J.119 Yang. G. Mussawisade. Gompper. 58002 McWhirter. M. (2008). G. 016706-1 . S. Exact solution of the Bernoulli matching model of sequence alignment Journal of Statistical Mechanics : Theory and Experiment. Salerno. I. Karevski. Kinetics and dynamics of wormlike micelles under shear Europhysics Letters. Elgeti.B.L. G. G. P. R. P09007 Ripoll. 293 . 20 (2008).-G. Kovats.C. S. G. V. V. M.. Schütz. Cooperation of Sperm in Two Dimensions: Synchronization... Y. 404209 Schütz. G. 78 (2008). G. Launois. Fullerene-cubane: X-ray Scattering Experiments and Monte Carlo Simulations Fullerenes Nanotubes and Carbon Nanostructures. Cambedouzou.016706-12 Popkov. 168302-1 . B. 81 (2008). M.. 78 (2008)... Schütz. Gompper. K.. Brandaut.. 253 (2008). M..H. 128 (2008). Lettinga. Attraction and Aggregation through Hydrodynamic Interactions Physical Review E. Michel. Gompper. D. J.. M.. Gompper. Phase behavior of a simple dipolar fluid under shear flow in an electric field Journal of Chemical Physics. R. Mechanical properties of icosahedral virus capsids Journal of Computer-Aided Materials Design. G. Molecular traffic control for a cracking reaction Journal of Catalysis. Vliegenthart... 011122 Priezzhev.. *.. H. J.. Goerigk.*. C. 3930 . J. S.S. H. E. Frielinghaus. G. Shi. 87 (2007) 3/5. 48 (2007). O. M. G. Wischnewski. 224508-1 . Ohl. G. Ziegenhagen.Institute of Solid State Research Neutron Scattering (IFF) 2007 Allgaier. U. 310 (2007). L. H. Willbold. A. S.*. Zorn.s263 Buchenau. Synthesis of Hydrophobic Poly(alkylene oxide)s and Amphiphilic Poly(alkylene oxide) Block Copolymers Macromolecules. Nonionic Surfactants with Linear and Branched Hydrocarbon Tails: Compositional Analysis. Ohl. H.*.* Neutron scattering evidence on the nature of the boson peak Journal of Physics: Condensed Matter. S.*. Kim. Koza..297 Feygenson. E. H.*. O.. Kentzinger. Z. A.*. 518 . T. J. M.. E. M. 295 . Deppe.. K. Geibel. Wischnewski. Jeon. Momono. Varga.*. 127 (2007). Steglich.6535 Frick. M. and Film Properties in Bicontinuous Microemulsions Langmuir.*. A.*.* When low. V.. 23 (2007) 12.*... Phase Behavior. T. R. Perez.*. K. Goerigk.3934 Faulhaber. Wang. 40 (2007) 3. Claesson. N.. 141 (2007) Frielinghaus. Patthey.. Contrast variation by anomalous X-ray scattering applied to investigation of the interface morphology in a giant magnetoresistance Fe/Cr/Fe trilayer Journal of Applied Crystallography. M. Tjernberg. 19 (2007). Biological systems as nanoreactors: Anomalouls small-angle scattering study of the CdS nanoparticle formation in multilamellar vesicles Journal of Physical Chemistry B. Varga. A. Chang.*. N. J. Y.. 75 (2007). A..*. Huber.*.*. Mudry. A.*.*. Dielectric and thermal relaxation in the energy landscape Philosophical Magazine. 6526 . Brückel. Mesot. Vesicles as reactors of nanoparticles: an anomalous small-angle X-ray scattering study of the domains rich in copper ions Journal of Applied Crystallography.*.and high-energy electronic responses meet in in cuprate superconductors Physical Review B. Zorn. Small-angle scattering model for multilamellar vesicles Physical Review E. Pailhés. Allgaier. Schweins. 76 (2007). Schmalzl.*. C. M.154908-8 143 .. Monkenbusch.1915 Bóta.* Probing the extent of the Sr2+ ion condensation to anionic polyacrylate coils: A quantitative anomalous small-angle x-ray scattering study Journal of Chemical Physics. Editorial European Physical Journal Special Topics: ST.. C. T. B. U.*. 051603-1 051603-8 Goerigk.*.*. Rücker. Ido.224508-6 De Luca.* Spatial separation of antiferromagnetism and superconductivity in CeCu2Si2 Journal of Magnetism and Magnetic Materials. Goerigk.*. E. U.. 40 (2007) 3. Loewenhaupt.. J. G. M. R. S. s259 . 205106 Buchenau.525 Bóta. F. Voigt. Stockert..* Neutron spin echo study of the dynamics of micellar solutions of randomly sulphonated polystyrene Polymer. M. 1911 . R. Prast.*.. 111 (2007) 8. Oda.538 Frank. Waigh.*.*. J. T. M. 532 . M. 154908-1 . Schnyder. M.*. 389 . Månsson.*.400 Chang. 40 (2007). Z. Jeevan.*.. Fabiani. A.. 22 (2007). Walter.3)Mn1-xFex)O3 (0 kleiner/gleich x kleiner/gleich 0. M. 111 (2007) 9.*. 184401 Holderer. H..*.N. K. D. Brückel. Monkenbusch. Skowronek. R. Lund. R. P. P.. H. Lindner.*. M.I. Willner. T.161 Iatrou.*..2) colossal magentoresistive manganites: Neutron spin-echo measurements Physical Review B. Wischnewski. Bodnarchuk. D.*. Stellbrink. H. 127 (2007)..A. M.*. Frielinghaus. Mueller. de Rosa.* Low energy excitations in CeBiPt Journal of Magnetism and Magnetic Materials. Richter. O. Shalguev. N. Hadjichristidis.s331 Maccarrone.. Monkenbusch. Evidence for two disparate spin dynamic regimes within Fe-substituted La0. C.*. K. Meuffels. Perßon. 1 (2007). Barandiarán.* Unraveling the equilibrium chain exchange kinetics of polymeric micelles using small-angle neutron scattering archtectural and topological effects Journal of Applied Crystallography. 094504 Klose. J. Allgaier. Die Megawatt-Spallationsneutronenquelle SNS: Neue Chancen für die Erforschung der kondensierten Materie Physik Journal. Prager..*.. Brückel. R. 222 (2007). 23 (2007) 19. 416 Ioffe. J. R..D. A. 19 (2007). O. H. H. H..*. J. Triolo. Fouquet. G. D'Errico. Perßon. Yoshino. J. Ikkala.803 Li. R. 799 . Willner.*.*.. V. 108 . Richter. N. M.. Dynamic properties of RecA protein filaments from E-coli and P-aeruginosa investigated by neutron spin echo Biofizika. Frielinghaus. D.016003-12 Lund. M. M. S. Takabatake. J. P.. Temperature-dependent crystal structure analysis of methyl iodide by high-resolution neutron powder spectroscopy Zeitschrift für Kristallographie. Buchsteiner. M.*. T. Larmor labeling by time-gradient magnetic fields Physica B: Condensed Matter. F.*. G.. R. L.* Architecturally induced multiresponsive vesicles from well-defined polypeptides. R.2173 144 . F. Richter. Ruokolainen. Bussmann.111 Kentzinger.*. Su.. T.. Radulescu. Correlation between structural and magnetic properties of La7/8Sr1/8Mn1-gammaO3 delta with controlled nonstoichiometry Journal of Physics: Condensed Matter. J. L. J. Zaccarelli..* Mesoscopic and Microscopic Investigation on Poly(vinyl alcohol) Hydrogels in the Presence of Sodium Decylsulfate Journal of Physical Chemistry B. Prager. F.. L.. M. Grimm. Richter. M. Quasielastic neutron scattering experiments including activation energies and mathematical modeling of methyl halide dynamics Journal of Chemical Physics.. V.*.46 Kirstein. Lindener.*. 52 (2007) 5. Frielinghaus.. Lo Celso. Romano... Iatrou..*. B.*.. D. 041503 Lebedev. M. 176226-1 -176226-12 Li.*. 016003-1 . E.*..*.* SANS Study of Polymer-Linked Droplets Langmuir. 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