Mixed Bilayers Research Articles

Electric Field Driven Changes of a Gramicidin Containing Lipid Bilayer Supported on a Au(111) Surface

Langmuir-Blodgett and Langmuir-Schaeffer methods were employed to deposit a mixed bilayer consisting of 90% of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 10% of gramicidin (GD), a short 15 residue ion channel forming peptide, onto a Au(111) electrode surface. This architecture allowed us to investigate the effect of the electrostatic potential applied to the electrode on the orientation and conformation of DMPC molecules in the bilayer containing the ion channel. The charge density data were determined from chronocoulometry experiments. The electric field and the potential across the membrane were determined through the use of charge density curves. The magnitudes of potentials across the gold-supported biomimetic membrane were comparable to the transmembrane potential acting on a natural membrane. The information regarding the orientation and conformation of DMPC and GD molecules in the bilayer was obtained from photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) measurements. The results show that the bilayer is adsorbed, in direct contact with the metal surface, when the potential across the interface is more positive than -0.4 V and is lifted from the gold surface when the potential across the interface is more negative than -0.4 V. This change in the state of the bilayer has a significant impact on the orientation and conformation of the phospholipid and gramicidin molecules. The potential induced changes in the membrane containing peptide were compared to the changes in the structure of the pure DMPC bilayer determined in earlier studies.

Spontaneous Buckling of Lipid Bilayer and Vesicle Budding Induced by Antimicrobial Peptide Magainin 2: A Coarse-Grained Simulation Study

Molecular mechanisms of the action of antimicrobial peptides on bacterial membranes were studied by large scale coarse-grained simulations of magainin 2-dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol (DPPC/POPG) mixed bilayer systems with spatial extents up to 0.1 μm containing up to 1600 peptides. Equilibrium simulations exhibit disordered toroidal pores stabilized by peptides. However, when a layer of peptides is placed near the lipid head groups on one side of the bilayer only, their incorporation leads to a spontaneous buckling of the bilayer. This buckling is followed by the formation of a quasi-spherical vesicular bud connected to the bilayer by a narrow neck. The mean curvature of the budding region is consistent with what is expected based on the dependence of the area per lipid on the peptide-to-lipid ratio in equilibrium simulations. Our simulations suggest that the incorporation of antimicrobial peptides on the exterior surface of a vesicle or a bacterial cell leads to buckling and vesicle budding, presumably accompanied by nucleations of giant transient pores of sizes that are much larger than indicated by equilibrium measurements and simulations.

Interactive Visual Analysis of Temporal Cluster Structures

AbstractCluster analysis is a useful method which reveals underlying structures and relations of items after grouping them into clusters. In the case of temporal data, clusters are defined over time intervals where they usually exhibit structural changes. Conventional cluster analysis does not provide sufficient methods to analyze these structural changes, which are, however, crucial in the interpretation and evaluation of temporal clusters. In this paper, we present two novel and interactive visualization techniques that enable users to explore and interpret the structural changes of temporal clusters. We introduce the temporal cluster view, which visualizes the structural quality of a number of temporal clusters, and temporal signatures, which represents the structure of clusters over time. We discuss how these views are utilized to understand the temporal evolution of clusters. We evaluate the proposed techniques in the cluster analysis of mixed lipid bilayers.

Non-ideal mixing and fluid–fluid immiscibility in phosphatidic acid–phosphatidylethanolamine mixed bilayers

Differential scanning calorimetry (DSC) was used to study the miscibility of phosphatidic acids (PAs) with phosphatidylethanolamines (PEs) as a function of chain length (n=14, 16) and degree of ionization of PAs at pH 4, pH 7, and pH 12. Phase diagrams were constructed using temperature data for onset and end of the phase transition obtained from the direct simulation of the heat-capacity curves. The phase diagrams were analyzed by simulations of the coexistence curves utilizing a four-parameter regular solution model. For PA-PE mixtures, the non-ideality parameters are a function of composition indicating non-symmetric non-ideal mixing behavior. At pH 7, where the PA component is negatively charged, the systems DMPA:DMPE and DPPA:DPPE have positive non-ideality parameters ρ (1) in both phases, indicating a preferred aggregation of like molecules. In contrast, DMPA:DPPE and DPPA:DMPE mixtures had negative ρ (1) values. Measurements at pH 4 showed that mixed pair formation is favored when PA is protonated. At pH 12 where PA is doubly charged, highly positive ρ (l1) parameters are obtained for the liquid-crystalline phase except for the system DPPA:DPPE (ρ (1)<0). This indicates clustering of like molecules and possibly domain formation in the liquid-crystalline phase. DPPA:DMPE at pH 12 even shows a miscibility gap in the liquid-crystalline phase. Obviously, despite the presence of doubly charged PA a fluid-fluid immiscibility is induced.

Self-Assembly of Mixed Anionic and Nonionic Surfactants in Aqueous Solution

We present the phase diagram and the microstructure of the binary surfactant mixture of AOT and C(12)E(4) in D(2)O as characterized by surface tension and small angle neutron scattering. The micellar region is considerably extended in composition and concentration compared to that observed for the pure surfactant systems, and two types of aggregates are formed. Spherical micelles are present for AOT-rich composition, whereas cylindrical micelles with a mean length between 80 and 300 Å are present in the nonionic-rich region. The size of the micelles depends on both concentration and molar ratio of the surfactant mixtures. At higher concentration, a swollen lamellar phase is formed, where electrostatic repulsions dominate over the Helfrich interaction in the mixed bilayers. At intermediate concentrations, a mixed micellar/lamellar phase exists.

The energetic and topography changes of mixed lipid bilayers deposited on glass

Characterization of the changes in wetting, energetic and topography of mixed phospholipid/sterol bilayer (BL) deposited on solid support was undertaken in these studies. Synthetic phospholipid (1,2-dipalmitoyl- sn-glycero-3-phosphocholine – DPPC) and sterol (cholesterol – Chol) were used. The lipid and mixed phospholipid/sterol bilayers were deposited on glass surface from chloroform solutions by spontaneous spreading or using Langmuir–Schaefer technique. The studied bilayers prepared by solution spreading were additionally incubated in water and then analyzed. Wettability of the films was investigated via contact angle measurements performed for three probe liquids (water, formamide and diiodomethane). The contact angles allowed evaluation of the total surface free energy and its components for particular systems applying two theoretical approaches, the one proposed by van Oss et al. (LWAB) and the other one of Chibowski (CAH). In order to visualize the changes of structure and properties of studied films their topography images were taken employing atomic force microscopy (AFM). The performed studies showed, that incorporation of cholesterol into the DPPC bilayers influenced their energetic and wetting properties, what reflected in the changes of contact angle and consequently the surface free energy of these films. The increasing sterol concentration fosters creation of rafts and organization of DPPC hydrocarbon chains on the layer surface. This causes changes in the surface free energy of mixed DPPC/Chol layers depending on the cholesterol concentration. The changes in topography of the studied layers observed with a help of AFM correlate well with the changes of their energetic properties.

Lipid Membranes with a Majority of Cholesterol: Applications to the Ocular Lens and Aquaporin 0

Using molecular dynamics (MD) simulations, we studied the structure and dynamics of two dimyristoylphosphatidylcholine (DMPC):cholesterol bilayers at concentrations representative of the ocular lens (ratios of 1:1 and 1:2). These MD simulations agree well with experimental deuterium order parameters and bilayer peak-to-peak distances. Although it is known that the average surface area per lipid rapidly decreases from low to moderate levels of cholesterol, our simulations indicate that there is a relatively small change in the average lipid area from 50 to 66.7% cholesterol (40.5 ± 0.2 and 39.5 ± 0.1 Å(2)/lipid, respectively). Radial distribution functions for the hydroxyl group on cholesterol indicate the formation of cholesterol-only nanoscale domains for the membrane with 66.7% cholesterol but a uniform distribution of cholesterol and DMPC for the membrane with 50% cholesterol. These small domains form a single shell of hexagonally packed cholesterols that are interconnected in a web-like structure of cholesterol. Calculations of internal DMPC dynamics show that the relaxation times for carbon-hydrogen reorientation of choline decrease with an increase in cholesterol, but the main body (carbonyl-glycerol to C11) is independent of cholesterol concentration. MD simulations of the aquaporin 0 tetramer show stabilization in its interactions with lipid membranes containing cholesterol by forming ring-ring stacking between surface aromatic residues of the protein and the rings of cholesterol. Moreover, there is an increase in hydrogen bonds with longer lifetimes in a mixed bilayer of DMPC and cholesterol.

Self-consistent mean-field model for palmitoyloleoylphosphatidylcholine-palmitoyl sphingomyelin-cholesterol lipid bilayers.

The connection between membrane inhomogeneity and the structural basis of lipid rafts has sparked interest in the lateral organization of model lipid bilayers of two and three components. In an effort to investigate anisotropic lipid distribution in mixed bilayers, a self-consistent mean-field theoretical model is applied to palmitoyloleoylphosphatidylcholine (POPC)--palmitoyl sphingomyelin (PSM)--cholesterol mixtures. The compositional dependence of lateral organization in these mixtures is mapped onto a ternary plot. The model utilizes molecular dynamics simulations to estimate interaction parameters and to construct chain conformation libraries. We find that at some concentration ratios the bilayers separate spatially into regions of higher and lower chain order coinciding with areas enriched with PSM and POPC, respectively. To examine the effect of the asymmetric chain structure of POPC on bilayer lateral inhomogeneity, we consider POPC-lipid interactions with and without angular dependence. Results are compared with experimental data and with results from a similar model for mixtures of dioleoylphosphatidylcholine, steroyl sphingomyelin, and cholesterol.

Molecular Binding of Black Tea Theaflavins to Biological Membranes: Relationship to Bioactivities

Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavins with membranes was explored by simulating the interactions of three theaflavin molecules (theaflavin, theaflavin-3-gallate, and theaflavin-3,3'-digallate) with a mixed bilayer composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). The simulations show that the theaflavins evaluated have an affinity for the lipid bilayer surface via hydrogen bonding. The molecular structure of theaflavins influenced their configuration when binding to the bilayer surface, as well as their ability to form hydrogen bonds with the lipid headgroups. The theaflavin-bilayer interactions studied here help to define structure-function relationships of the theaflavins and provide a better understanding of the role of theaflavins in biological processes. The significance of the results are discussed in the context of black tea composition and bioactivity.

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers.

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.

Polyunsaturated and Saturated Phospholipids in Mixed Bilayers: A Study from the Molecular Scale to the Lateral Lipid Organization

Polyunsaturated and Saturated Phospholipids in Mixed Bilayers: A Study from the Molecular Scale to the Lateral Lipid Organization

Gap Junction Hemichannel Interactions with Zwitterionic Lipid, Anionic Lipid, and Cholesterol: Molecular Simulation Studies

Interactions with membrane lipids can exert dramatic functional consequences on gap junction proteins. Recent experimental work has highlighted the importance of anionic lipids and cholesterol in facilitating channel activity. In this work, we have employed a coarse-grained molecular model in conjunction with molecular dynamics (MD) simulations to study the interactions between a connexin 26 (Cx26) hemichannel and a number of lipid species, including palmitoyloleoylphosphatidylcholine (POPC), anionic palmitoyloleoylphosphatidic acid (POPA), and cholesterol, in order to identify sites at the protein interface which may exhibit preferential, specific binding to these lipids, as well as determine the characteristics of these interactions. We have also employed an atomistic model of Cx26 embedded in a mixed PA/PC bilayer as a comparison and to elucidate further lipid-protein interactions. Our simulation results suggest enrichment of interfacial PA at the intracellular leaflet at high bulk PA concentrations. PC can form tight binding interactions with the hemichannel, particularly at intersubunit crevices (classical nonannular sites). In mixed bilayers, however, POPA competes with POPC for these sites, displacing the latter in some cases. While the residues responsible for interactions with PC and PA are similar, the latter exhibits a unique property of being capable of forming stable hydrophilic contacts with multiple residues spanning two different adjacent subunits at both leaflets of the bilayer, as opposed to POPC which can only do so at the extracellular side. These results suggest that POPA may be essential to channel function by acting as an intersubunit lipid bridge. Additionally, we propose that the compositional enrichment of POPA at the Cx26 interface may serve important roles in voltage gating. Simulation of a mixed POPC:cholesterol bilayer suggests that the hemichannel enhances the transbilayer mobility of vicinal cholesterols, increasing the likelihood of site-hopping and interleaflet flip-flop transitions.

Molecular Dynamics Studies of Ion Permeation in VDAC

The voltage dependent anion channel (VDAC) in the outer membrane of mitochondria serves an essential role in transport of metabolites and electrolytes between the cell matrix and mitochondria. To examine its structure, dynamics, and mechanisms underlying its electrophysiological properties, we have performed a total of 1.77 μs molecular dynamics simulations of human VDAC isoform 1 in DOPE/DOPC mixed bilayers in 1M KCl solution with transmembrane potentials of 0, ±25, ±50, ±75, and ±100 mV. The calculated conductance and ion selectivity are in good agreement with the experimental measurements. In addition, ion density distributions inside the channel reveal possible pathways for different ion species. Based on these observations, a mechanism underlying the anion selectivity is proposed; both ion species are transported across the channel, but the rate for K+ is smaller than that of Cl- because of the attractive interactions between K+ and residues on the channel wall. This difference leads to the anion selectivity of VDAC.

Molecular Dynamics Studies of Ion Permeation in VDAC

The voltage-dependent anion channel (VDAC) in the outer membrane of mitochondria serves an essential role in the transport of metabolites and electrolytes between the cell matrix and mitochondria. To examine its structure, dynamics, and the mechanisms underlying its electrophysiological properties, we performed a total of 1.77 μs molecular dynamics simulations of human VDAC isoform 1 in DOPE/DOPC mixed bilayers in 1 M KCl solution with transmembrane potentials of 0, ±25, ±50, ±75, and ±100 mV. The calculated conductance and ion selectivity are in good agreement with the experimental measurements. In addition, ion density distributions inside the channel reveal possible pathways for different ion species. Based on these observations, a mechanism underlying the anion selectivity is proposed; both ion species are transported across the channel, but the rate for K+ is smaller than that for Cl− because of the attractive interactions between K+ and residues on the channel wall. This difference leads to the anion selectivity of VDAC.

The Fukutin Transmembrane Domain: Capturing the Complexity of the Golgi Apparatus Membrane via Multiscale MD Simulations

The membrane of the Golgi apparatus is a complex mixture of lipids and proteins. In the present work we describe multiscale molecular dynamics simulations of transmembrane protein domains in model membranes that represent the in vivo Golgi environment. The transmembrane domain of glycosyltransferases is required for their correct sorting within the Golgi apparatus. The hydrophobic thickness and oligomerization state of the transmembrane domains have been proposed to mediate this sorting. Fukutin, is a putative Golgi glycosyltransferase implicated in muscular dystrophy.We employ atomistic and coarse-grained molecular dynamics simulations to investigate the stability and membrane interactions of the fukutin transmembrane domain, using various models of the membrane. Our atomistic simulations reveal that the fukutin transmembrane domain can exist as a stable α helix irrespective of the headgroup charge, fatty acid saturation or hydrophobic thickness of the lipid bilayer. Coarse-grained simulations reveal that the tilt angle of the fukutin transmembrane domain is highly variable and dependent upon its local environment; both the hydrophobic thickness and the headgroup charge of the lipid bilayer can alter the tilt angle of the protein. Lastly, we study the dynamics of the fukutin transmembrane domain in a mixed lipid bilayer whose composition closely mimics the complex lipid headgroup composition of the Golgi apparatus.

A simulation-Based Test of the Constant Membrane Fluidity of Different Mixed Lipid Bilayers at Different Temperatures

It is in the textbook that cells change the lipid composition to maintain the constant membrane fluidity in response to environmental changes. For example, in E. coli, the ratio of the saturated fatty acids is increased as the temperature increases, and that of the unsaturated fatty acids is increased as the temperature decreases to keep the constant fluidity of membrane bilayers.(Noriaki Katsui, Tetsuaki Tsuchido, Mitsuo Takano, and Isao Shibasaki, 1981) The aim of this work is to investigate the membrane fluidity. We have performed molecular dynamic simulations of twenty lipid systems with pure palmitoyloleoylphosphatidylcholine(POPC), pure dioleoylphosphatidylcholine(DOPC), pure dipalmitoylphosphatidylcholine(DPPC), mixtures of DOPC and DPPC, and mixtures of DOPC, DPPC, and POPC. These systems were simulated at four different temperatures: 283K, 293K, 303K, and 313K. The ratios of lipids in mixed bilayer systems differ according to their temperatures. We ran three independent runs for each system at each temperature to improve the conformational sampling and the statistics of the results. We will present various membrane properties, such as chain order parameters, membrane thickness, per-lipid areas, compressibility, for each system in each temperature, and discuss the results in terms of the membrane constant fluidity.

Refining and Testing CHARMM Lipid Parameters for Biologically Important Membranes

Biological membranes form a barrier to protect the cell from its environment and consist of a wide variety of lipids. A recent modification to the CHARMM lipid force filed, CHARMM36 (C36), resulted in significant improvements in deuterium order parameters (SCD), water hydration, and area compressiblities. Moreover, C36 simulations resulted in excellent surface areas per lipid compared to experiment with NPT molecular dynamics (MD). We have extended this force field to aliphatic chain branched lipids, which exists in many bacterial membranes. MD simulations of DPhPC agree with experimental form factors and suggest that current lipid force field parameters are valid for these branched chain lipids. Branching leads to an increase in average surface areas per lipid, area elastic moduli and lipid axial relaxation times. However, MD simulations of mixed bilayers with cholesterol and polyunsaturated lipids suggest that the atomistic force field describing these lipids requires modification (improper sterol orientation and head-to-head group spacings). MD simulations on decalin resulted in a heat of vaporization that is 10 kJ/mol lower than experiment, but modifications to the Lennard-Jones parameters of cholesterol yielded near perfect agreement with experiment. Simulations with these parameters were in excellent agreement with experimental SCDs for the DMPC acyl chain and SCDs for ring and chain positions on cholesterol. Moreover, these simulations agree favorably with experimental form factors of DMPC-cholesterol bilayers. However, simulations with a polyunsaturated lipid, DAPC, and cholesterol still resulted in the abovementioned inaccuracies. Since the thickness of these polyunsaturated bilayers was too large by ∼8 A, high-level quantum mechanical (QM) calculations were used for dihedrals adjacent to the double bonds. It was found that the previous force field restricted the conformational flexibility and MD simulations are underway to test the QM-based potentials accuracy compared to experimental diffraction studies.

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH(2)) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC-dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d(54)-DMPC) and mixed d(54)-DMPC-DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC-DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by (31)P NMR. However, no perturbations in the T(1) relaxation nor the T(2-) values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).

Interactions of the Antimicrobial Peptide Maculatin 1.1 and Analogues with Phospholipid Bilayers

The interactions of the antimicrobial peptide, maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH2) and two analogues, with model phospholipid membranes have been studied using solid-state NMR and circular dichroism spectroscopy. Maculatin 1.1 and the P15G and P15A analogues displayed minimal secondary structure in water, but with zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles displayed a significant increase in α-helical content. In mixed phospholipid vesicles of DMPC and anionic dimyristoylphosphatidylglycerol (DMPG), each peptide was highly structured with ~80% α-helical content. In DMPC vesicles, the native peptide displayed moderate head group interaction and significant perturbation of the lipid acyl chains. In DMPC/DMPG vesicles, maculatin 1.1 promoted formation of a DMPG-enriched phase and moderately increased disorder towards acyl chain ends of DMPC in the mixed bilayer. Both analogues showed reduced phospholipid head group interactions with DMPC but displayed significant interactions with the mixed lipid system. These effects support the preferential activity of these antimicrobial peptides for bacterial membranes.

Structure and order of DODAB bilayers modulated by dicationic gemini surfactants

Cationic liposomes have been extensively studied from the experimental and theoretical standpoints, motivated both by their fundamental interest and by potential applications in drug delivery and gene therapy. However, a detailed understanding of the nature of interactions within mixed bilayers containing cationic gemini surfactants is still lacking. This work focuses on the structural and dynamic properties of DODAB membranes in the presence of dicationic gemini surfactants. A thermodynamic characterization of the phase transitions in the mixed systems has been carried out by differential scanning calorimetry, while insight into the molecular interactions in the bilayer has been provided by molecular dynamics. For this purpose, variations in the gemini spacer and tail length, as well as in the respective molar fraction, have been included in both experimental and simulation studies. The results indicate that the influence of cationic gemini surfactants upon the thermotropic behavior and degree of order of DODAB structures is controlled by a complex interplay between charge density, conformation and hydrophobic effects, for which a detailed rationale is provided.