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The Size of a Reverse Micelle
Wednesday, March 2, 2016 spanning the hydrophobic slab. Importantly, an unbiased (lipidþsolvent only) free energy landscape for poration can be obtained by reweighting. Preliminary tests suggest that a similar ‘stalk device’ can also direct stalk formation in the same manner. 2820-Pos Board B197 Mitochondrial Membrane Fusion: Computational Modeling of Mitofusins Dario De Vecchis, Antoine Taly, Marc Baaden, Je´roˆme He´nin. Laboratoire de Biochimie The´orique UPR 9080 CNRS, Institut de Biologie Physico Chimique, PARIS, France. Fzo1, a large GTPase of the Dynamin-Related Proteins superfamily, is a key component in mitochondrial outer membrane fusion and is required for maintaining mitochondrial dynamics and morphology. The protein is anchored to the outer membrane by two transmembrane segments and its N-terminal GTPase domain and C-terminal are exposed to the cytosol. Recent data indicate that the GTPase domain of Fzo1 would induce a conformational change concomitant with mitochondrial tethering, thus promoting membrane fusion [1]. We investigate the structure and dynamics of Fzo1 through molecular modeling and all-atom simulation in a model mixed lipid bilayer, closely linked to experiments. Our structural model integrates information from several template structures, experimental knowledge, as well as ab initio models of the transmembrane segments that are unique to Fzo1. The model is validated experimentally through charge swap mutations across predicted salt bridges and a series of N-terminal truncation mutants indicates that this region is dispensable for function. Our approach unravels hinges domains involved in the conformational change and identified critical residues required for protein stability. Moreover several point mutation found to disrupt the architecture of the protein are located in the coiled-coil domain which has been shown fundamental for the protein [2]. Finally, we dissected key residues in protein-GDP interaction providing fundamental insights about molecular mechanisms by which mitofusins catalyze membrane fusion. [1] Cohen, M.M., Amiott, E.A., Day, A.R., Leboucher, G.P., Erin, N.P., Glickman, M.H., McCaffery, J.M, Shaw, J.M. and Weissman, A.M. (2011). J. Cell Sci. 124, 1403-1410. [2] Griffin, E. E. and Chan, D. C. (2006). J. Biol. Chem. 281, 16599-16606. 2821-Pos Board B198 Molecular Dynamics Simulations of Membrane Translocation of Dendrimers Valreia Marquez-Miranda1,2, Ingrid Araya-Duran1, Jeffrey Comer3, Maria Carolina Otero Acuna4, Jonathon Canan5, Fernando Danilo Gonzalez Nilo1. 1 Center for Bioinformatics and Integrative Biology (CBIB), Universidad Andres Bello, Santiago, Chile, 2Fraunhofer Chile Research, Fraunhofer chile research, Chile, 3Institute of Computational Comparative Medicine, Nanotechnology Innovation Center of Kansas State, Kansas State University, Manhattan, KS, USA, 4Center for Integrative Medicine and Innovative Science (CIMIS), Universidad Andres Bello, Santiago, Chile, 5Fraunhofer Chile Resarch, Santiago, Chile. Dendrimers are a class of hyperbranched polymer whose structure and surface chemistry can be precisely controlled, which makes them suitable for applications in nanomedicine, such as delivery of therapeutics. Terminal groups of dendrimers have been shown to influence the rate of internalization into cells. For instance, previous articles have demonstrated that dendrimers PAMAMNH2, PAMAM-OH and PAMAM-COOH of generation 4 (G4) can be taken up by A549 lung cells, but at varying rates, depending on the nature of the terminal groups. It was concluded that amine-terminated dendrimers are more rapidly internalized, since they are prone to interact with negative groups of the cell membrane, such as sialic acid. Several studies have addressed the phenomenon of dendrimer translocation across membranes using computer simulations. However, these studies are challenging and largely infeasible, due to the slow diffusion of lipids composing the membranes. In the present study, we take advantage of coarse-grained models based on MARTINI force field, which have been successfully employed to assess membrane binding of proteins. Furthermore, a highly mobile membrane mimetic, HMMM based on a biphasic solvent model was also employed, to compare the behavior of dendrimers in full-atom and a coarse grained system. Both models permit us to study the translocation of new G3-guanidine, G3-NH2 and G3-OH dendrimers on a reasonable time scale. We expect to delineate why some dendrimers have faster rates of translocation and why some produce membrane perturbations that induce cytotoxicity. These results will further the development of dendrimers as carriers of therapeutic molecules.
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2822-Pos Board B199 The Size of a Reverse Micelle Gozde Eskici1, Paul H. Axelsen2. 1 Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA, 2Pharmacology, Biochemistry, and Biophysics, University of Pennsylvania, Philadelphia, PA, USA. Reverse micelles (RMs) are nanometer sized water droplets, surrounded by surfactant and immersed in an organic solvent. They are used to encapsulate molecules for studies of structure and reactivity. The water droplets in RMs have a narrow size distribution that depends on the water:surfactant ratio (i.e. the water loading ratio, Wo). Various measurements of their size have been reported, but they are inconsistent - most likely due to the models and assumptions required to interpret the measurements. We hypothesized that RMs have a narrow size distribution because - for any given Wo - there is one size that yields a minimum overall interaction energy per surfactant (i.e. the minimum chemical potential). Therefore, we undertook a study of RM size using all-atom molecular dynamics simulations, and AOT reverse micelles in isooctane, all having Wo=7.5 but with varying size. We found that the interaction energy per surfactant molecule was dominated by electrostatic interactions between anionic AOT head groups, sodium cations, and water. The interaction energy per surfactant at Wo=7.5 exhibits a clear minimum when there are 62 AOT molecules per reverse micelle, which agrees with estimates in published light scattering studies. When fewer AOT are in each reverse micelle, the attractive forces between anionic AOT head groups and sodium cations are reduced due to the physical separation of cations and anions by hydration shells, as well as the dielectric effect of the hydration shells. When there are more AOT in each reverse micelle, the repulsive forces between AOT anions become dominant. Consequently, reverse micelles that are too small or too large for any given Wo value have excess internal energy that drives mass exchange and changes in RM shape. Our results provide fundamental insight into the basis for the size and narrow size distribution of RMs. 2823-Pos Board B200 Fluctuating Lipid Nanodomains Near Critical Transitions George R. Heath, Stephen D. Evans, Simon D. Connell. Physics and Astronomy, University of Leeds, Leeds, United Kingdom. Lipid organization in cellular membranes is fundamental to many cellular processes, and in recent years the hypothesis of lipid ‘‘rafts’’ has grown from one of simple macroscopic phase co-existance to a more dynamic description, encompassing modulated phases, compositional fluctuations and micro-emulsions. The static and dynamic behaviour of critical lipid mixtures have been characterised by fluorescence microscopy of GUV’s, but at length scales of >1 mm due to optical resolution limits. Here we present High-Speed AFM data of the critical phase behaviour of a model cell, imaging with nanometre lateral resolution and at up to 8 frames per second. Below the critical temperature (in the macroscopic phase separation regime) the boundaries of micron-sized domains were observed fluctuating with amplitudes of 10’s of nm’s. This motion has been quantitatively analysed to give line tensions in the range 1 pN (similar to that observed in GUV’s), reducing linearly with temperature down to a low of 0.05 pN close to the critical point. As temperature is increased whilst imaging we show the crossing of the critical temperature (TC) resulting in the breakdown of large domains into much smaller nanoscale fluctuations. The fluctuations at and above TC result in unstable 5 - 100 nm domains that have a lifetime of seconds to almost 1 minute, again dependent upon temperature. Domains persist up to 10 degrees above TC. For direct comparison with our results we perform simple Monte Carlo 2D Ising model simulations which show closely analogous behaviour for line tensions, phase structure break down and domain lifetimes. Our results give the first direct insight into membrane dynamics at the nanoscale. 2824-Pos Board B201 Micelles and Bicelles as Membrane Mimics for Membrane Protein Characterization Ashton Brock1, Shelby Lipes1, Ryan Oliver1, Svetlana Baoukina2, Peter Tieleman2, Linda Columbus1. 1 Chemistry, University of Virginia, Charlottesville, VA, USA, 2Biological Sciences, University of Calgary, Calgary, AB, Canada. Micelles and bicelles are used to isolate membrane proteins for biophysical characterization, functional assays, and high-resolution structure determination. Utilizing these membrane mimics requires an extensive screening process to identify the appropriate amphiphilic environment to stabilize a functional membrane protein. To enable rational mimic selection, the goal of this research is to investigate the structural properties of micelles and bicelles to determine
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2822-Pos Board B199 The Size of a Reverse Micelle Gozde Eskici1, Paul H. Axelsen2. 1 Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA, 2Pharmacology, Biochemistry, and Biophysics, University of Pennsylvania, Philadelphia, PA, USA. Reverse micelles (RMs) are nanometer sized water droplets, surrounded by surfactant and immersed in an organic solvent. They are used to encapsulate molecules for studies of structure and reactivity. The water droplets in RMs have a narrow size distribution that depends on the water:surfactant ratio (i.e. the water loading ratio, Wo). Various measurements of their size have been reported, but they are inconsistent - most likely due to the models and assumptions required to interpret the measurements. We hypothesized that RMs have a narrow size distribution because - for any given Wo - there is one size that yields a minimum overall interaction energy per surfactant (i.e. the minimum chemical potential). Therefore, we undertook a study of RM size using all-atom molecular dynamics simulations, and AOT reverse micelles in isooctane, all having Wo=7.5 but with varying size. We found that the interaction energy per surfactant molecule was dominated by electrostatic interactions between anionic AOT head groups, sodium cations, and water. The interaction energy per surfactant at Wo=7.5 exhibits a clear minimum when there are 62 AOT molecules per reverse micelle, which agrees with estimates in published light scattering studies. When fewer AOT are in each reverse micelle, the attractive forces between anionic AOT head groups and sodium cations are reduced due to the physical separation of cations and anions by hydration shells, as well as the dielectric effect of the hydration shells. When there are more AOT in each reverse micelle, the repulsive forces between AOT anions become dominant. Consequently, reverse micelles that are too small or too large for any given Wo value have excess internal energy that drives mass exchange and changes in RM shape. Our results provide fundamental insight into the basis for the size and narrow size distribution of RMs. 2823-Pos Board B200 Fluctuating Lipid Nanodomains Near Critical Transitions George R. Heath, Stephen D. Evans, Simon D. Connell. Physics and Astronomy, University of Leeds, Leeds, United Kingdom. Lipid organization in cellular membranes is fundamental to many cellular processes, and in recent years the hypothesis of lipid ‘‘rafts’’ has grown from one of simple macroscopic phase co-existance to a more dynamic description, encompassing modulated phases, compositional fluctuations and micro-emulsions. The static and dynamic behaviour of critical lipid mixtures have been characterised by fluorescence microscopy of GUV’s, but at length scales of >1 mm due to optical resolution limits. Here we present High-Speed AFM data of the critical phase behaviour of a model cell, imaging with nanometre lateral resolution and at up to 8 frames per second. Below the critical temperature (in the macroscopic phase separation regime) the boundaries of micron-sized domains were observed fluctuating with amplitudes of 10’s of nm’s. This motion has been quantitatively analysed to give line tensions in the range 1 pN (similar to that observed in GUV’s), reducing linearly with temperature down to a low of 0.05 pN close to the critical point. As temperature is increased whilst imaging we show the crossing of the critical temperature (TC) resulting in the breakdown of large domains into much smaller nanoscale fluctuations. The fluctuations at and above TC result in unstable 5 - 100 nm domains that have a lifetime of seconds to almost 1 minute, again dependent upon temperature. Domains persist up to 10 degrees above TC. For direct comparison with our results we perform simple Monte Carlo 2D Ising model simulations which show closely analogous behaviour for line tensions, phase structure break down and domain lifetimes. Our results give the first direct insight into membrane dynamics at the nanoscale. 2824-Pos Board B201 Micelles and Bicelles as Membrane Mimics for Membrane Protein Characterization Ashton Brock1, Shelby Lipes1, Ryan Oliver1, Svetlana Baoukina2, Peter Tieleman2, Linda Columbus1. 1 Chemistry, University of Virginia, Charlottesville, VA, USA, 2Biological Sciences, University of Calgary, Calgary, AB, Canada. Micelles and bicelles are used to isolate membrane proteins for biophysical characterization, functional assays, and high-resolution structure determination. Utilizing these membrane mimics requires an extensive screening process to identify the appropriate amphiphilic environment to stabilize a functional membrane protein. To enable rational mimic selection, the goal of this research is to investigate the structural properties of micelles and bicelles to determine