Session P29: Biomolecular Structure and Functions

Interactions between model bacterial membranes and synthetic antimicrobials.

Antimicrobial peptides comprise a key component of innate immunity for a wide range of multicellular organisms. It has been shown that natural antimicrobial peptides and their analogs can permeate bacterial membranes selectively. There are a number of proposed models for this action, but the detailed molecular mechanism of the induced membrane permeation remains unclear. We investigate interactions between model bacterial membranes and a prototypical family of phenylene ethynylene-based antimicrobials with controllable hydrophilic and hydrophobic volume fractions, controllable charge placement. Preliminary results from synchrotron small angle x-ray scattering (SAXS) results will be presented.   

Observation of membrane fusion between individual virus particles and supported lipid bilayers

A portion of the host cell membrane is incorporated into newly produced, enveloped virus particles during an active infection. Fusion of that viral membrane with the membrane of targeted host cells is generally accepted to be a key step for the infection of normal cells as a virus spreads among a normal cell population. For the best studied enveloped viruses, viral proteins catalyze the membrane fusion reaction during a low pH step along the cellular endocytotic pathway. To gain a better understanding of the molecular mechanisms underlying viral membrane fusion, we have constructed an \textit{in vitro} fluorescence assay to allow high resolution, real time measurements of Sindbis viral fusion to supported lipid bilayers. Single particle tracking is used to observe individual virus particles. The mixing of a fluorescent dye incorporated into the viral membrane with the supported bilayer reports fusion. We present results regarding the effects of different lipid blends as well as different buffer conditions on membrane fusion for Sindbis virus. We compare the fusion of virus produced in mammalian cells to that from insect cells. .   

Simulating Domain Formation and Fusion in Lipid Bilayers

The lipid dynamics is the source of the variety of membrane structures and dynamics found in celss. Many of the interesting phenomena of biomembranes involve time scales of at least microseconds, which have been beyond simulations until recently. Coarse-grained models of lipid molecules have been developed to reach these long time scales and maintain the essential physical character of the molecules. Using these models in molecular dynamics simulations, time scales in the $\mu$s to ms range are treatable. As examples, simulations of domain formation and membrane fusion will be presented. In mixed lipid systems, the formation of domains is now understood to be an active component in biological processes. Our simulations reveal the dynamics of the lipid molecules that form domains in binary systems. In particular, the correlation between the two monolayers of the bilayer is dependent on the molecular structure of the lipid molecules. Membrane fusion is a fundamental process of cellular transport and infection processes. Understanding the basic principles governing membrane fusion has many important consequences. The coarse-grained molecular dynamics simulations show how lipid molecular structure influences the fusion process.   

Lipid Coupling in Asymmetric Supported Lipid Bilayers Revealed by Fluorescence Correlation Spectroscopy

In biological systems, phospholipids asymmetry in two leaflets is a key feature of cell membranes for membrane biogenesis, intracellular fusion and signal transduction. Detailed information of the interactions and dynamics of the asymmetric membranes is paramount for design of applications. Here we use fluorescence correlation spectroscopy (FCS) to measure the coupling between 1, 2-dilauroyl-\textit{sn}-glycero-3-phosphocholine (DLPC) and 1, 2-dipalmitoyl-\textit{sn}-glycero-3-phosphocholine (DPPC) in asymmetric planar-supported bilayers (PSLBs), at temperatures where DLPC is in the fluid phase but DPPC is in the gel phase. Asymmetric PSLBs were prepared by placing dilute fluorescent-labeled 1, 2-dimeristoyl-\textit{sn}-glycero-3-phosphoethanolamine (DMPE) in DLPC leaflet as the probe for measuring lateral diffusion within the host leaflet environment. By constructing asymmetric bilayers where DLPC is alternatively in the top and in the bottom leaflet, we compare lipid coupling between the two leaflets with frictional interaction between the leaflets and the nanometer-thick water layer that separates the bottom leaflet from the solid support.   

Dissipative Particle Dynamics Simulation of Structure Properties of Lipid Micelles

We chose dissipative particle dynamics (DPD) simulation to study the micelle structure properties. The self-assembly lipid is modeled by a flexible chain with head and tail particles. By changing interaction parameters between the solution particles and the lipid particles, three types of solution are considered: water, oil and water-oil mixture. It is found that the tail/head chains forming the micelle core have a very disorder distribution. The relation between the core radius with the aggregation number as well as the number of oil particles inside is obtained based on the simulation results and the molecular packing parameter. The mean density of particles in the core is higher than that in the simulation box. For micelles without oils inside, the density depends on the number of head particles in a lipid molecule, and the dependence becomes weaker as more oil particles are captured. At the core surface, head particles form clusters with water particles incorporated between the clusters. Comparisons with results from other simulation method such as MD show that DPD achieves high performance in simulating micelle formation and structure properties.   

Anomalously Slow Domain Growth in Membranes with Asymmetric Transbilayer Lipid Distribution

The effect of asymmetry in transbilayer lipid distribution on the phase separation of self-assembled multicomponent fluid vesicles is investigated numerically via dissipative particle dynamics. We show that this asymmetry induces a spontaneous curvature wich alters significantly the morphology and dynamics of the lipid mixture. In particular, at intermediate tension, domain growth is found to be anomalously slow dynamics. In contrast, in the limiting cases of low and high tensions, the dynamics proceed toward full phase separation.   

Molecular simulation studies of tail-length effects in mixed-lipid bilayers

Because lipid lateral diffusion is slow on the time-scale accessible to atomistic molecular dynamics (MD) simulations, equilibrium clustering and segregation in mixed-lipid bilayers are impractical to study through conventional computational approaches. A hybrid MD-Monte Carlo method employing lipid mutation moves within the semi-grand canonical ensemble method has been implemented for mixtures of lipids of differing tail lengths. For DLPC:DPPC mixtures, equilibration is demonstrated during simulation runs nearly two orders of magnitude shorter than estimates for standard MD. Statistical measures of lateral association in bilayer slabs and of partitioning among membrane micro-environments (flat bilayers, inner and outer leaflets of curved bilayers, and bilayer edge) will be presented.   

Stabilization of Model Membrane Systems by Disaccharides. Quasielastic Neutron Scattering Experiments and Atomistic Simulations

Trehalose, a disaccharide of glucose, is often used for the stabilization of cell membranes in the absence of water. This work studies the effects of trehalose on model membrane systems as they undergo a melting transition using a combination of experimental methods and atomistic molecular simulations. Quasielastic neutron scattering experiments on selectively deuterated samples provide the incoherent dynamic structure over a wide time range. Elastic scans probing the lipid tail dynamics display clear evidence of a main melting transition that is significantly lowered in the presence of trehalose. Lipid headgroup mobility is considerably restricted at high temperatures and directly associated with the dynamics of the sugar in the mixture. Molecular simulations provide a detailed overview of the dynamics and their spatial and time dependence. The combined simulation and experimental methodology offers a unique, molecular view of the physics of systems commonly employed in cryopreservation and lyophilization processes.   

Protein crystals on phase-separating model membranes

We study the interplay between the crystallization of proteins tethered to membranes and separation within the membranes of giant unilamellar vesicles (GUVs) composed of DOPC, sphingomyelin (SM), and cholesterol. These model membranes phase separate into coexisting liquid domains below a miscibility transition temperature. This phase separation captures some aspects of the formation of lipid rafts in cell membranes and demonstrates the influence of membrane composition on raft formation. Real cell membranes have a much more complicated structure. There are additional physical constraints present in cell membranes, such as their attachment to the cytoskeleton and the presence of membrane bound proteins. The self-association of membrane proteins can influence the membrane phase behavior. We begin to investigate these effects on model tethered protein- loaded membranes by incorporating a small amount of biotin-X- DPPE into our GUVs. The biotinylated lipid partitions into a cholesterol-poor phase; thus, streptavidin binds preferentially to one of the membrane phases. As streptavidin assembles to form crystalline domains, it restricts the membrane mobility. We examine the effect of this protein association on lipid phase separation, as well as the effect of the lipid phase separation on the crystallization of the tethered proteins.   

Model systems to investigate the effect of cholesterol on the transfection efficiency of lipoplexes

Motivated by its important role in lipid-mediated gene delivery, we have studied the effect of cholesterol on membrane fusion. While recent work in our group has identified the membrane charge density as a critical parameter for transfection efficiency (TE) of lamellar, DOPC containing cationic lipid-DNA (CL-DNA) complexes [1-3], this model cannot fully explain the effect of cholesterol, suggesting that a different mechanism is responsible for the observed enhancement of TE. A model system using negatively charged giant vesicles has been developed to mimic the interaction of the cell membrane with CL-DNA complexes containing cholesterol. Differences in fusogenic properties have been observed as a function of the amount of cholesterol present in the CL-DNA complexes, and a fluorescence resonance energy transfer based assay was employed to quantify this effect. X-ray diffraction confirms that the lamellar structure seen with CL- DNA complexes is retained with the addition of cholesterol. Funding provided by NIH GM-59288 and NSF DMR-0503347. [1] A.J. Lin et al, \textit{Biophys. J.}, 2003, V84:3307-3316. [2] K. Ewert et al, \textit{J. Med. Chem.}, 2002, V45:5023-5029. [3] A. Ahmad et al., \textit{J. Gene Med., }2005, V7:739-748.   

Had a drink last night? How alcohol interacts with biological membranes

We have performed extensive 100 ns molecular dynamics simulations to study the effect of methanol and ethanol on two different lipid bilayer systems (POPC and DPPC) in the fluid phase at 323 K [1,2]. We studied both structural changes induced by the alcohols and the dynamics of the system. It turned out that ethanol was able to penetrate the membranes whereas methanol was not able to do so. In particular, ethanol prefers to be accommodated in the vicinity of the lipid headgroup region. We also determined the dependence of lipid chain ordering on ethanol concentration and quite surprisingly found that to be non-monotonous. We explain that in terms of modified surface tension [2]. Finally, we determined lifetime of hydrogen bonds to be about 1 ns and found that be in excellent agreement with NMR results. \newline \newline [1] B.W. Lee, et al, Fluid Phase Equilibria 225, 63-68 (2004) \newline [2] M. Patra et al, Biophys. J., in press 2005   

Solid domain rafts in lipid vesicles and scars

The free energy of a crystalline domain coexisting with a liquid phase on a spherical vesicle may be approximated by an elastic or stretching energy and a line tension term. The stretching energy generally grows as the area of the domain, while the line tension term grows with its perimeter. We show that if the crystalline domain contains defect arrays consisting of finite length grain boundaries of dislocations (scars) the stretching energy grows linearly with a characteristic length of the crystalline domain. We show that this result is critical to understand the existence of solid domains in lipid-bilayers in the strongly segregated two phase region even for small relative area coverages. The domains evolve from caps to stripes that become thinner as the line tension is decreased. We also discuss the implications of the results for other experimental.   

Efficient calculation of mechanical properties of multicomponent phospholipid bilayers with Monte Carlo simulations

We present a systematic study of the mechanical properties of multicomponent phospholipid bilayers. Two sets of systems are considered. The first consists of a mixture of DioleoylPhosphatidylethanolamine (DOPE) and DioleoylPhosphatidylcholine (DOPC) phospholipids. These two molecules have different head groups but the same chain length. The second system consists of a mixture of DilauroylPhosphatidylcholine (DLPC) and DistearoylPhosphatidylcholine (DSPC); these molecules have different chain lengths but the same head group. We use atomistic and coarse grain models, coupled to advanced Monte Carlo simulation techniques, to examine the structure and mechanical properties of the bilayers. Our results for pure systems are in quantitative agreement with experiment. Experimental data for mixed bilayers are not available, but our results indicate that their mechanical behavior is highly non-linear, a finding that we can interpret in terms of the composition and the resulting structure of the mixtures.   

Roughness effect on vesicle adhesion characterised by a novel micropipette-based technique

Numerous biological processes have to go through a cell adhesion process, which make the fundamental study of the adhesion of cells on solid substrate a key research topic in cellular biophysics. We will present our work on the adhesion of a single vesicle on a substrate. A vesicle is held at the end of a micropipette mounted on a micromanipulator and put into contact with a surface. We developed a novel technique to directly measure adhesion using the spring-constant of an L-shaped micropipette when pulling the vesicle from the substrate. The substrate is made of a micropatterned polymer film coated with a thin layer of gold to promote adhesion with the vesicle. The effect of the surface roughness can therefore be carefully characterized.   

Structure of Cholesterol Helical Ribbons, Self-Assembling Biological Springs.

Helical ribbons with characteristic pitch angles form spontaneously in a variety of quaternary surfactant-lipid-sterol-water solutions. These helical ribbons form in a variety of axial lengths, widths and radii. Surprisingly, however, they all have pitch angles of either 11 or 54\r{ }. Our X-ray diffraction studies of individual ribbons confirm that the remarkable stability of each of the two pitch angles is related to a crystalline nature of the ribbons. The small size (of 100 x 10 x 0.1 $\mu $m$^{3})$ and the significant curvature of the ribbons produce weak and broad Bragg peaks. Therefore, novel methods are used to analyze these data. The structure of these ribbons is similar to that of cholesterol monohydrate. Interestingly, there is an evidence for a superlattice structure, resembling that found in thick films of cholesterol grown at the air-water interface.