Mechanistic Insights into Membrane Bending by Protein Crowding: Understanding the Role of Membrane Composition, Phase Separation and Free Energy of Protein Binding

Wednesday, March 2, 2016 prostate cancer cells showed that exosomes are particularly enriched in longchain SM (Biochim. Biophys.Acta, 2013, 1831, 1302–1309). In this study, we use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on these data. The simulations show interdigitation of the long-chain SM to the opposite leaflet, which is quite expected, but interestingly we found out that interdigitation was particularly strong in asymmetric bilayers compared to symmetric ones. We observed that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. We further showed that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet. 2859-Pos Board B236 Transbilayer Registration of Liquid-Ordered Domains: No Interactions at the Membrane Midplane Required Timur R. Galimzyanov1,2, Veronika V. Alexandrova2, Peter Pohl3, Sergey A. Akimov1,2. 1 Bioelectrochemistry, Frumkin Institute of Physical Chemistry and Electrochemistry of RAS, Moscow, Russian Federation, 2Department of Theoretical Physics and Quantum Technologies, National University of Science and Technology ‘‘MISiS’’, Moscow, Russian Federation, 3Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria. The mechanism responsible for the registration of liquid-ordered (Lo) domains in the two membrane leaflets is a matter of debate. As an alternative to the thus far enigmatic interactions at the membrane midplane, we propose that minimization of the line tension around the thicker Lo bilayer drives registration. Based on the continuum elasticity theory, we demonstrate that the thickness mismatch at the Lo/Ld boundary results in elastic deformations of lipid molecules. The deformations require a minimum of energy FD when the domain boundaries are shifted relative to each other by several nanometers1. Accumulation of lipids or peptides with non-zero spontaneous curvature in the thin rim around the domains further sharply decreases FD, thus explaining the line activity of monosialoganglioside GM1. In addition to FD, the mutual attraction of stiffed membrane regions must contribute to domain registration2. Since energy FU is required to prevent membranes from undulating, maximizing the membrane area that is free to undulate by aligning the stiff Lo domains from opposing leaflets minimizes FU. Thus, domain registration minimizes FU and FD explaining why (i) transbilayer coupling can occur without interaction at the membrane midplane and (ii) registration does not require specific lipids in Lo domains. The work was supported by the Russian Foundation for Basic Research (15-5415006) and by the Austrian Science Fund (I12267). 1) Galimzyanov, Molotkovsky, Bozdaganyan, Cohen, Pohl, Akimov. Elastic Membrane Deformations Govern Interleaflet Coupling of Lipid-Ordered Domains. Phys.Rev.Lett. 115:088101, 2015. 2) Horner, Antonenko, Pohl. Coupled diffusion of peripherally bound peptides along the outer and inner membrane leaflets. Biophys.J. 96:2689-2695, 2009. 2860-Pos Board B237 N-RAS Lipid Anchor Adsorption to Membranes as a Function of Lipid Composition and Curvature Jannik B. Larsen1, Celeste Kennard2, Søren L. Pedersen3, Knud J. Jensen3, Nikos S. Hatzakis1, Mark J. Uline2, Dimitrios Stamou1. 1 Chemistry, Bionanotechnology and Nanomedicine Laboratory, Nano-Science Center, Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark, 2 Chemical Engineering, University of South Carolina, Columbia, SC, USA, 3 Chemistry, Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark. Protein recruitment to biological membranes is motivated by either highly selective recognition of specific target membrane components or non-specific attraction to general physical properties of the membrane, such as charge, lipid heterogeneity, and curvature. Here we discuss the interaction between lipid-anchored proteins and lipid membranes from a comprehensive examination of how features of the membrane and its lipid constituents, including lipid head-group size, composition, heterogeneity, membrane thickness, degree of unsaturation, and membrane geometry, effect the adsorption ability of the proteins. Of key importance is the strong interconnection among these compositional and morphological elements in mediating the binding of peripheral membrane proteins. As a model protein, we use the dual lipidated (palmitoyl and farnesyl) anchoring motif of the signaling GTPase N-Ras (tN-Ras). We find marked augmentation in tN-Ras adsorption with increasing degree of membrane curvature—a trend that is tightly regulated by the bilayer characteristics mentioned above. Experimental results are fully reproduced by a

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molecular level theoretical model of the systems under study. Of note, the theory suggests an explicit dependence on the lateral pressure profile of the membrane’s hydrophobic region to be the mechanism and cause of variation in protein density with membrane curvature and composition. Relief in the lateral pressure of the bilayer’s outer leaf, upon its expansion induced by increasing curvature, reduces the work requirement for lipidanchor insertion into the membrane. Furthermore, the inherent pressure profile of the hydrophobic channel, at a given curvature, is unique with regard to membrane composition, which allows for fine-tuning of lipidated-protein density. 2861-Pos Board B238 Segmentation of Membrane Protein Motion in the Axon Initial Segment Christian M. Winterflood1, David Albrecht2, Gabrielle de Wit3, Philipp Kukura3, Helge Ewers4. 1 Randall Division of Cell and Molecular Biophysics, King’s College London, London, United Kingdom, 2Institute of Biochemistry, ETH Zu¨rich, Zu¨rich, Switzerland, 3Physical and Theoretical Chemistry Laboratory, Oxford University, Oxford, United Kingdom, 4Institute of Chemistry and Biochemistry, Freie Universita¨t Berlin, Berlin, Germany. The axon initial segment (AIS) is a structure rich in specific cytoskeletal molecules that play important roles in the concentration of ion-channels that are required for action-potential generation. The establishment of a postulated diffusion barrier to the lateral exchange of membrane molecules in the AIS correlates with the enrichment of specific cytoskeletal molecules at this structure during development. Recently, a repetitive pattern of actin, spectrin and ankyrin forming ring-like structures perpendicular to the direction of axonal propagation has been discovered, that is interconnected via spectrin tetramers. This structure may finally provide the long sought direct physical correlate to the diffusion barrier at the AIS. Here, we perform repeated high-throughput single-molecule tracking on individual live primary hippocampal neurons during AIS development (DIV 3 - 10). We furthermore analyze the lateral mobility of lipid-anchored and transmembrane molecules with microsecond tracking at a resolution of few nanometers via interferometric scattering (iSCAT). Finally, we correlate the lateral motion of membrane molecules to the organization of the AIS cytoskeleton. We find that the lateral motion of membrane molecules becomes reduced in the AIS during development and that this reduction correlates with cytoskeletal organization into ring-like structures. The lateral motion of membrane molecules in the AIS plasma membrane is locally confined to awithin a repetitive pattern of 190 nm spaced segments along the AIS axis, consistent with the observed spacing of the cytoskeletal rings. Our data provide mechanistic insight into the diffusion barrier function in of the AIS. 2862-Pos Board B239 Mechanistic Insights into Membrane Bending by Protein Crowding: Understanding the Role of Membrane Composition, Phase Separation and Free Energy of Protein Binding Gokul Raghunath, Brian Dyer. Department of Chemistry, Emory University, Atlanta, GA, USA. Morphological changes in lipid membranes are hallmarks of a number of cellular processes like sorting, transport, etc. The dense crowding of the membrane environment with proteins and receptors has motivated many thorough academic investigations into the effects of macromolecular crowding on membrane surfaces. A number of recent studies have indicated that membrane reshaping could be driven by steric pressure between proteins co-localized on membrane domains. While these studies provide conclusive evidence for the membrane bending process, a detailed physical and mechanistic basis for this phenomenon is lacking. We provide a thermodynamic picture for this phenomenon through Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC) and fluorescence microscopy using Ni-Nitrilotriacetic (NTA) acid and His-Tag interaction as a model system. Using ITC, we observe almost an order of magnitude increase in binding affinity for NTAfunctionalized liposomes that display gel-fluid phase coexistence, as opposed to homogenous fluid compositions. This elevated affinity could be eliminated by thermal phase transition from gel-fluid to fluid, highlighting the importance of phase separation in modulating the strength of this binding interaction. DSC revealed that protein binding modulates the long-range lipid order substantially. In conjunction with the complicated nature of the binding isotherm, the DSC results indicate that the protein-binding event is coupled to a secondary exothermic process, presumably due to membrane deformation. Further ITC and fluorescence microscopy experiments reveal that the formation of

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Wednesday, March 2, 2016

membrane tubules upon protein binding is sensitive to membrane composition, phase separation and free energy of protein binding. Taken together with a predictive theoretical model that is currently under development, we believe that these results significantly advance our current understanding of the thermodynamics of membrane bending by protein crowding. 2863-Pos Board B240 A Detergent-Free Approach to Membrane Protein Research: PolymerBounded ‘‘Native’’ Nanodiscs Jonas M. Do¨rr1, Juan J. Dominguez Pardo1, Marleen H. van Coevorden-Hameete2, Stefan Scheidelaar1, Martijn C. Koorengevel1, Casper C. Hoogenraad2, J. Antoinette Killian1. 1 Chemistry, Utrecht University, Utrecht, Netherlands, 2Biology, Utrecht University, Utrecht, Netherlands. Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alternative to detergents for applications in membrane research [1]. Most notably, these amphipathic polymers can be used to directly extract and purify membrane proteins from intact cells of different organisms in the form of ‘‘native nanodiscs’’. These particles stabilize the protein in a near native environment comprising conserved native lipids as well as other membrane components and they readily allow for structural and functional characterization of the protein [2,3]. To evaluate the general applicability of SMA-mediated membrane protein solubilization, we employed a combined imaging and biochemistry approach using HeLa cells as a model. The results indicate that SMA solubilization of (human) cells is an all-or-none process that is not specific for any (sub) cellular membrane, as seen by the solubilization of all intracellular organelles that were tested. These findings suggest that SMA isolation is applicable to any membrane protein irrespective of which cellular membrane it resides in. Since lipid properties strongly influence the solubilization process [4], we then tested whether SMA exhibits selectivity for certain lipids within a given membrane. To this end, we studied the effect of the polymer on model membranes with different lipid compositions. The results revealed a promiscuity of SMA with respect to lipid headgroups in homogeneous lipid mixtures. However, in phase-separating systems of fluid phases with either gel-phase or liquidordered phases it showed a distinct preference for lipids in the fluid phase. Implications for the solubilization of proteins from such membranes will be discussed. [1] Do¨rr JM, et al., 2015, Eur Biophys J, submitted. [2] Swainsbury DJK, et al., 2014, Angew Chem Int Ed Engl 53(44):1180311807. [3] Do¨rr JM, et al., 2014, PNAS 111(52):18607-18612. [4] Scheidelaar S, et al., 2015, Biophys J 108(2):279-290. 2864-Pos Board B241 ‘‘Anionic H-Bonds’’ Structure Two Simple Bilayers, One Natural Edward G. Hohenstein1, Michael A. Green1, Alisher Kariev1, Saranga Naganathan2, Mary Manning Cleveland2, Thomas H. Haines1,3. 1 Chemistry, City College of City University of New York, New York, NY, USA, 2Danica Foundation, New York, NY, USA, 3Chemical Biology and Signal Receptors, Rockefeller University, New York, NY, USA. ‘‘Anionic H-Bonds’’ Structure Two Simple Bilayers, One Natural Living membranes consist of bilayers of primarily anionic polar lipids. We describe two simple, single-chain lipid bilayers: pure oleate C18D9 carboxylate, and a chlorosulfolipid (CSL), a C24, hexachloro-1,14-disulfate, from the fresh water (pH 4.3) alga, Ochromonas danica. Oleate bilayers are formed either from Na-oleate or from micelles of oleic acid. Both monolayer headgroups, carboxylate and sulfate, trap a HD between their oxyanion pairs, (‘‘anionic H-bond’’). In an aprotic medium they have strong H-bonds (<=20 kcal/ bond), analogous to the maleate anion (JACS 2015, 137, 5730). The maleate anionic H-bond is stabilized by ring strain; here van der Waals interactions between the thin methylene chains, together with the bulk of both headgroups, compacts oxyanions forming the anionic H-bonds. O. danica’s CSLs’ sulfates, at C1 and C14, form two sulfate sheets, one at C1 between headgroup pairs and the other at C14 in the low dielectric domain. There aren’t other lipids nor proteins in the O. danica surface bilayers. Thus each bilayer has four sulfate sheets, two at C1,C14. Computations show that chloro groups bond hydronium ions at near covalent bond strength in a hydrophobic domain. Inside each monolayer, the C14-sulfate sheet creates a negative field deep in the bilayer. Two chloros are on C2 adjacent to the C1-sulfate surface. They attract hydronium ions to the surface bilayer, each brings water and Hþ to the C14 sulfate sheet. Water stabilizes the sulfate sheet and HD forms anionic H-bonds between sulfates. These four strong sheets protect O. danica from osmotic bursting as do walls in prokaryotes.

2865-Pos Board B242 Curvature-Induced Lipid Sorting in Plasma Membrane Tethers Svetlana Baoukina1, Helgi I. Ingolfsson2, Siewert J. Marrink2, D. Peter Tieleman1. 1 University of Calgary, Calgary, AB, Canada, 2University of Groningen, Groningen, Netherlands. Membrane tethers are nanotubes formed by lipid bilayers. They are efficient structures for cellular transport and communication, and for storage of excess membrane area. Previous tether pulling experiments provided insights on membrane mechanical properties, and the curvature effects on phase behavior and distribution of coexisting phases. However, detailed information on tether properties and variations in composition is challenging to obtain experimentally due to the small diameters and dynamic nature of tethers. Here we provide a molecular view on curvature-induced lipid sorting in plasma membrane tethers. We pulled tethers from an idealized plasma membrane model using molecular dynamics simulations with the coarse-grained Martini model. The membrane consists of 63 lipid types with an asymmetric distribution of components between the leaflets [JACS, 2014, 136, 14554]. The tethers are formed by applying an external constant force to a lipid patch in the direction normal to the bilayer plane [Biophys J, 1012, 102, 1866]. Pulling is performed both from the inner and outer leaflets, corresponding to the direction in and out of the cell, respectively. As a result of pulling, we observe redistribution of different lipid types along the regions of different curvature without macroscopic phase separation. Depending on the direction of pulling, the distribution of lipids and the tether properties differ. 2866-Pos Board B243 Direct Observation of Ordered and Disordered Membrane Domains in B Cell Plasma Membranes using Multi-Color Super-Resolution Fluorescence Microscopy and Application to B Cell Receptor Signaling Matthew B. Stone, Sarah Shelby, Marcos Nunez, Sarah Veatch. Biophysics, University of Michigan, Ann Arbor, MI, USA. Many immune receptors are hypothesized to be correlated with domains of unique membrane composition, sometimes termed ‘‘lipid rafts,’’ which modulate activity of the receptor during the immune response. This compositional heterogeneity is hypothesized to be analogous to liquid ordered/ liquid disordered (lo/ld) phase separation observed in giant unilamellar vesicles and in vesicles harvested from the plasma membrane. However, their existence and behavior has been difficult to measure directly due to the small size of these domains and the small difference between the composition of the domain and the rest of the plasma membrane. Here, we utilize multi-color super-resolution microscopy (STORM and PALM) to quantitate the local density of various ld or lo preferring membrane probes around B cell receptor (BCR) clusters. We show in control measurements that ld or lo preferring membrane probes have differential partitioning around clusters of proteins having strong phase preference. Clusters of lo preferring cholera toxin subunit B are enriched in lo probe and depleted of ld probe. Inversely, clusters of an ld preferring transmembrane peptide are depleted of lo probe and enriched in ld probe. We apply this technique to BCR, which is thought to anchor a raft domain during antigen binding. We find that BCR clusters in chemically fixed and live cells are enriched in lo probe and depleted of ld probe, indicating a lo-like composition is anchored around BCR clusters. We find that this anchored domain is sensitive to the ambient temperature and receptor phosphorylation. These experiments also quantitate the contribution of membrane composition on the interaction between Lyn and the BCR. These results show that lipidmediated forces can play important roles in organizing proteins during signaling processes. 2867-Pos Board B244 High Resolution Imaging Atomic Force Microscope Study of Interactions at the Membrane-Fluid Interface Chiara Rotella1,2, Jason I. Kilpatrick1, Simona Capponi1,2, Miguel Holmgren3, Francisco Bezanilla4, Eduardo Perozo4, Suzanne P. Jarvis1,2. 1 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland, 2School of Physics, University College Dublin, Dublin, Ireland, 3Molecular Neurophysiology Section, Porter Neuroscience Research Center, NINDS, National Institutes of Health, Bethesda, MD, USA, 4Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA. The cell membrane is essential for all living systems, serving as a barrier between cells and their environment. It is typically composed of a lipid bilayer, containing embedded and/or anchored proteins that mediate different biological function such as energy conversion, signal transduction and solute transport [1]. To elucidate the basic structure of biological membranes it is