CHOL: a Molecular Dynamics Study

Tuesday, February 14, 2017

Membrane Structure III 1889-Pos Board B209 Liquid Membrane Fluctuations Drive Ordered Monolayer Domain Alignment and Raft Stacking Timur R. Galimzyanov1,2, Veronika V. Alexandrova1, Petr I. Kuzmin2, Peter Pohl3, Sergey A. Akimov2. 1 Theoretical Physics & Quantum Technologies, National University of Science and Technology, Moscow, Russian Federation, 2 Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Moscow, Russian Federation, 3Institute of Biophysics, Johannes Kepler University, Linz, Austria. We identify the mechanism of interbilayer and transbilayer coupling of liquidordered domains in lipid membranes. The driving force for such coupling has thus far remained unrevealed, yet membrane domains are always adopt bilayer conformation. Liquid-ordered domains also demonstrate transbilayer correlative behaviour by uniting into stacks in the multilamellar structures. Various hypothesis were put forward to explain coupling phenomena. Transbilayer coupling is usually explained by some phenomenological interaction between ordered and disordered domains in opposite monolayers. While interbilayer coupling is described by hydration forces that are supposed to be different for ordered and disordered phases. Both hypotheses lack sufficient evidence and theoretical basis, while such a widespread cooperative behaviour of rafts makes to suggest the existence of common driving force for these phenomena. We provide the universal mechanism of such striking behaviour. Using continuum elasticity theory, we show that elastic deformations alone are the driving force of ordered domain coupling. The coupling is provided by two independent mechanisms. The first one is due to membrane dynamic membrane shape fluctuations; the corresponding energy is proportional to domain area. This mechanism utilizes the idea that stiff regions in both monolayers attract each other because their registration minimizes spatial restraints on membrane undulations, i.e. domain registration maximizes entropy. The second mechanism bases on the domain boundary energy minimization; the coupling energy is proportional to domain boundary length. In the framework of thickness mismatch model we show that the minimum domain boundary energy is achieved in registered conformation, making monolayer domains and non-stacked configurations energetically unfavourable [1, 2]. Combination of these two mechanisms results in alignment driven by lipid deformations, the membranes fundamental property, which does not require introducing any specific features, like interactions at the membrane midplane, different hydration forces, etc. Fluctuation-based model is universal and does not depend on particular properties of lipid membrane and can be applied to any system, composed of elastic films with non-uniform elastic characteristics. 1. Galimzyanov et al., Physical Review Letters 115.8 (2015). 2. Galimzyanov et al., Physical Review Letters 116.7 (2016). 1890-Pos Board B210 Mechanical Properties of Model Erythrocytes Membranes from Healthy and Hereditary Spherocytosis Subjects Bruna R. Casadei1, Amanda C. Carita´1, Vale´ria F. Dutra2, Eneida de Paula3, Rumiana Dimova4, Maria S. Figueiredo2, Karin A. Riske1. 1 Biophysics, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2 Hematology and Hemotherapy, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 3Biochemistry, State University of Campinas, Campinas, Brazil, 4 Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Defects in the erythrocyte membrane can cause hereditary spherocytosis (HS), a congenital hemolytic anemia caused by a deficiency in cytoskeletal or integral proteins. To understand the complex behavior of the composing lipids and their involvement in the stability and organization of the membrane structure, we have used optical microscopy in giant unilamellar vesicles (GUVs) and assessed the membrane mechanical properties and the response of GUVs to Triton X-100. GUVs were composed by lipid extracted from the erythrocyte membranes of both healthy individuals (GUVs-NI) and HS (GUVs-HS) patients, as well as by purified lipids (palmitoyl oleoyl phosphatidylcholine, POPC, and POPC:sphingomyelin:cholesterol, 2:1:2). The bending rigidity of GUVs was quantified using fluctuation analysis. GUVs-HS were found to be softer than GUVs-NI and GUVs-2:1:2, probably reflecting differences in their acyl chain composition. The pore edge tension was measured from the dynamics of macropore resealing after application of electric pulses. The edge tension of erythrocyte lipids was shown to be lower than that of the biomimetic system (2:1:2), probably due to the presence of charged lipids. The solubiliza-

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tion profile of GUVs-NI, GUVs-HS and GUVs-2:1:2 were very similar: Triton X-100 first induced phase separation (liquid-ordered/liquid-disordered phase) followed by partial solubilization of the liquid-disordered phase. The results are discussed in terms of differences in lipid composition, as revealed from the fatty acid and headgroup composition analysis of the erythrocyte-lipid extracts. 1891-Pos Board B211 Lipids Regulated through Membrane Topography Revealed by Single Particle Tracking Xinxin Woodward, Abir Maarouf Kabbani, Christopher V. Kelly. Physics, Wayne State University, Detroit, MI, USA. The sorting of lipids and protein on plasma membrane are critical to the life of eukaryotic cells. For example, in the process of phagocytosis, phosphatidylinositol 4,5-bisphosphate (PIP2) accumulates around phagosomal cup and leads to actin remodeling. However the mechanisms of phagocytosis initiation is yet to be fully understood. We will be resolving these mechanisms via understanding the regulations of phospholipids such as PIP2 by membrane structure, i.e., studying the interplay between nanoscale curvature and PIP2 accumulation in curved supported lipid bilayers (SLBs). We hypothesize that PIP2 will be sorted based on membrane topography since membrane curvature has been shown to induce segregation of various lipids and proteins (Tian and Baumgart, Biophys. J., 2009). Since diffusion is a great indicator of membrane properties, the diffusion of lipids are quantified on nanoscale curvature and flat membrane by single particle tracking (SPT) paired with polarized localization microscopy (PLM). To create SLB with nanoscale curvature sites, giant unilamellar vesicles (GUV) were burst on 48 nm diameter nanoparticles over a coverslip substrate. Fluorophorescently tagged phospholipids were applied to the top leaflet after the formation of SLBs to minimize the membrane-substrate interaction. Single particle tracking (SPT) indicates that DPPE-Rhodamine diffuses 4 times faster on flat membrane than on 48 nm curvature sites. This result implies the membrane viscosity increase with curvature. In future experiments, TopFlourPIP2 will be introduced to SLB top leaflet with nanoscale curvature sites that are supported by nanoparticle of varying size. Diffusion and concentration of TopFlour-PIP2 and other phospholipids will be studied on flat membrane verses nanoscale curvature sites. Understanding the molecular sorting in lipid bilayers will reveal the mechanisms of PIP2 accumulation in nanoscale curvature initiation. 1892-Pos Board B212 Archaeal-Inspired Lipids Exhibit Low Membrane Permeability due to Entropic Effects Geoffray Leriche1, Karthikeyan Diraviyam2, Young Hun Kim1, Takaoki Koyanagi1, Olivia Eggenberger3, Thomas B.H. Schroeder3, Michael Mayer3, Jerry Yang1, David Sept2. 1 Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA, 2Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 3Adolphe Merkle Institut, Fribourg, Switzerland. Many archaea are able to withstand extremes of temperature, pressure, pH and/or levels of salt. One unique feature of these extremophiles that gives them this capability is that their membranes are made from covalently linked, tetraether lipids that form a monolayer rather than a traditional bilayer. Here we present experimental and computational results on a series of synthetic archaeal-inspired lipids, looking at their biophysical properties and rates of permeation. Apart from a number of interesting mechanical and material properties, we find that tethered lipids exhibit a much stronger dependence on the entropy of activation for small molecules to cross the membrane. In relating our measured permeation rates with properties of the membrane, we find that the order parameters for the lipid tails describe the changes in entropy, and the combination of area per lipid, membrane thickness and short-range diffusion coefficient can predict our permeation results. Interestingly, we also see that the rates of water penetration into the core of the membrane provide an excellent empirical measure that matches our experimental permeation rates. 1893-Pos Board B213 Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures of POPC/ CHOL: a Molecular Dynamics Study Fernando Favela-Rosales1, Arturo Galva´n-Herna´ndez2, Jorge Herna´ndez-Cobos2, Iva´n Ortega-Blake2. 1 Instituto Tecnolo´gico Superior Zacatecas Occidente, Sombrerete, Mexico, 2 Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Mexico. Phase diagrams on ternary and quaternary lipid mixtures showing the existence of liquid-disordered (ld), liquid-ordered (lo) and a mixed phase (ldþlo)

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are now well established. Although the ldþlo is generally accepted for binary mixtures of cholesterol and phospholipids with saturated acyl chains, the existence of a phase separation in mixtures of cholesterol with phospholipids that have one saturated and one unsaturated acyl chain is not universally accepted. There are reports of a phase diagram on the mixtures POPC/Chol and POPC/Erg and nystatin activity co-related to this purported phase diagram; with a maximal activity appearing in the mixed phase. In a recent work we presented a Molecular Dynamics (MD) study of a POPC membrane with different cholesterol concentrations along the phase diagram and computed the order parameters binned in the xyplane as well as the cholesterol density. The results support the existence of a lateral heterogeneity of cholesterol, present mainly in the mixed phase. Here we extend the previous study to consider also membrane thickness as well as the evolution of these distributions along a ms of simulation. We observed structures that support the idea of cholesterol lateral heterogeneity is present in this binary mixture. The theoretical distributions were compared to Atomic-Force Microscopy (AFM) images from studies of the same system on supported lipid bilayers. The combined study of AFM and MD simulations allowed us to advance in the understanding of the molecular structure changes resulting from phase differences. Funding: DGAPAPAPIIT-RG100416.

1896-Pos Board B216 Bending Modulus of Bolalipids under the U-Shapes Diffusion Sergei I. Mukhin, Daniyar Gabdullin. Theoretical Physics and Quantum Technologies Department, Moscow Institute for Steel and Alloys, Moscow, Russian Federation. We propose a phenomenological model to calculate frequency dispersion of the bending modulus of bolalipid membrane with lateral diffusive motion of small concentration of the bolalipid molecules in the U-shape conformation. A local curvature of the membrane causes an increase of the elastic energy, that could be released by dynamically induced U-shapes concentration imbalance on the opposing sides of the bolalipid layer. An effective attractive/repulsive potential acting upon the U-shapes on the opposite sides of the curved regions of the membrane are defined using the calculations of the local curvature induced by a difference of the U-shapes concentrations on the opposite sides of the membrane. When local spontaneous curvature caused by the U-shapes imbalance equals the local curvature induced by membrane bending the potential acting on the U-shapes turns to zero. Allowing for U-shapes lateral diffusion coefficient, the latter condition permits us to derive dynamic equations that describe in essence the dispersion of the bending modulus as function of the bending frequency. A possible comparison of the theoretical prediction with the proper experiment is discussed.

1894-Pos Board B214 Giant Vesicles Fabricated from Paper Anand B. Subramaniam. School of Engineering, University of California, Merced, Merced, CA, USA. Giant lipid vesicles are useful in vitro models for cells and for studying biochemical processes. Here we report a straightforward and widely accessible technique, PAPYRUS, Paper-Abetted liPid hYdRation in aqUeous Solutions, to produce cell-sized vesicles that are free of residual solvents and that are not exposed to electric fields during the growth process. We find that when placed in aqueous solutions, numerous giant vesicles formed from lipid films coated on cellulose fibers of filter and chromatography paper. Vesicles of different lipid compositions can be formed in various aqueous media such as ultrapure water, physiologically relevant ionic buffers, and sugar solutions. Fluorescent labeling and analysis demonstrated that cellulose does not incorporate into the membranes of the vesicles. The insolubility of cellulose in water, even at elevated temperatures, allows the formation of vesicles with lipids with high transition temperatures. The porosity of paper allows gentle fluid flow normal to the paper to free vesicles into the bulk. The extreme ease of the PAPYRUS technique, coupled with not requiring a power-source or function generator, allows massive parallelization using readily available or easily improvised fluid receptacles such as Eppendorf tubes or polystyrene multiwell plates.

1897-Pos Board B217 Different Static and Similar Kinetic Phase Behavior Seen for Monoolein and a Sister Compound Andrew D. Folkerts, Paul E. Harper. Department of Physics and Astronomy, Calvin College, Grand Rapids, MI, USA. Monoglycerides form a rich variety of non-lamellar phases, including minimal surface based cubic phases and the inverted hexagonal phase. Monoolein is a particularly popular monoglyceride that is widely utilized for drug encapsulation and protein crystallization. We use DSC (differential scanning calorimetry) to look at the static and kinetic phase behavior of a sister compound, mononoadecenoin (MNd), which differs from monoolein by the addition of a single methylene. Despite this small difference, the temperature of the cubic to inverted hexagonal phase transition is 30 C lower in MNd than in monoolein. We measure the hysteresis of this transition as function of temperature ramp rate and concentration of added sucrose solution. The hysteresis exhibits a power law dependence on the ramp rate. Interestingly, despite the difference in the phase transition temperatures, the power law exponent as function of temperature is the same for MNd and monoolein.

1895-Pos Board B215 Manipulation of Phase-Like Domains within Intact B Cell Plasma Membranes and Visualization of their Composition using Super-Resolution Microscopy Marcos F. Nunez, Matthew B. Stone, Sarah L. Veatch. Biophysics, University of Michigan, Ann Arbor, MI, USA. Protein sorting based on liquid-ordered (Lo) or liquid-disordered (Ld) phase preference is readily observed in giant plasma membrane vesicles (GPMVs) at low temperatures but is not as easily detected in the intact cells from which they are derived. Here we utilize two-color super-resolution microscopy in combination with cross-correlation analysis to quantify the sorting of two minimal inner leaflet anchored-peptides proximal to clusters of cholera toxin B subunit (CTxB) in chemically fixed CH27 B cells. We find that the local density of a Lo-partitioning peptide is increased while the local density of a Ld-partitioning peptide is reduced in the vicinity of CTxB clusters when compared to the average density of peptides on the cell surface. This in combination with additional results confirm cell plasma membranes can support lipid domains which resemble the Lo and Ld phases in GPMVs and span leaflets of the plasma membranes. Ongoing work is aimed at detecting heterogeneity in intact cells in the absence of artificial CTxB clustering, and at distinguishing possible physical bases of the observed lipid-mediated membrane heterogeneity in intact cells. These experiments are characterizing the changes in enrichment and depletion of phase-partitioning peptides with respect to CTxB upon changes in temperature and composition of the plasma membrane. These results will be compared to predictions of Ising model simulations to test for consistency between that model and the behavior of the phase-like domains we have observed in B cell plasma membranes.

1898-Pos Board B218 Hysteresis and the Cholesterol-Dependent Melting Transition of the Martini Model Alexis Webb, Clement Arnarez, Edward Lyman. University of Delaware, Newark, DE, USA. Extensive Martini simulation data, totaling 5 ms, is presented for binary mixtures of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol. Using simulations initiated from both gel and liquid-disordered (L d ) phases, significant and strongly cholesterol-dependent hysteresis in the enthalpy as a function of temperature is observed for cholesterol mole fractions from 0 to 20 mol% — adding just a single cholesterol to each leaflet of a pure DPPC bilayer more than doubles the width of the hysteresis loop relative to a pure DPPC bilayer. Although the precise phase transition temperature cannot be determined due to the hysteresis, the data are consistent with a first order gel to fluid transition, which increases in temperature with cholesterol. At 30 mol% cholesterol, no hysteresis is observed, and there is no evidence for a continuous transition, in either structural parameters like the area per lipid or in the heat capacity as a function of temperature. The results are consistent with a single uniform phase above a critical cholesterol composition between 20 and 30 mol% in Martini, while highlighting the importance and difficulty of obtaining the equilibrium averages to locate phase boundaries precisely in computational models of lipid bilayers. 1899-Pos Board B219 Ultradonut Topology of the Nuclear Envelope Mehdi Torbati1, Tanmay P. Lele2, Ashutosh Agrawal1. 1 University of Houston, Houston, TX, USA, 2University of Florida, Gainesville, FL, USA. Lipid membranes exhibit a variety of morphologies tailored to perform specific functions of cells and their organelles. A unique lipid structure is the nuclear envelope which houses the genome and plays a vital role in genome organization and signaling pathways. The nuclear envelope is composed of two fused membranes with thousands of toroid-shaped pores with extremely high