Binary Bilayers Research Articles

Comparative Computer Simulation Study of Cholesterol in Hydrated Unary and Binary Lipid Bilayers and in an Anhydrous Crystal

Models created with molecular dynamics simulations are used to compare the organization and dynamics of cholesterol (Chol) molecules in three different environments: (1) a hydrated pure Chol bilayer that models the Chol bilayer domain, which is a pure Chol domain embedded in the bulk membrane; (2) a 2-palmitoyl-3-oleoyl-d-glycerol-1-phosphorylcholine bilayer saturated with cholesterol (POPC-Chol50) that models the bulk membrane; (3) a Chol crystal. The computer model of the hydrated pure Chol bilayer is stable on the microsecond time scale. Some structural characteristics of Chol molecules in the Chol bilayer are similar to those in the POPC-Chol50 bilayer (e.g., tilt of Chol rings and chains), while others are similar to those in Chol crystals (e.g., surface area per Chol, bilayer thickness). The key result of this study is that the Chol bilayer has, unexpectedly, a dynamic structure, with Chol mobility similar to that in the POPC-Chol50 bilayer though slower. This is the major difference compared to Chol crystals, where Chol molecules are immobile. Also, water accessibility to Chol-OH groups in the Chol bilayer is not limited. On average, each Chol molecule makes 2.3 hydrogen bonds with water in the Chol bilayer, compared with 1.7 hydrogen bonds in the POPC-Col50 bilayer.

How does acyl chain length affect thermotropic phase behavior of saturated diacylphosphatidylcholine–cholesterol binary bilayers?

Thermotropic phase behavior of diacylphosphatidylcholine (CnPC)–cholesterol binary bilayers (n=14–16) was examined by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry. The former technique can detect structural changes of the bilayer in response to the changes in polarity around Prodan molecules partitioned in a relatively hydrophilic region of the bilayer, while the latter is sensitive to the conformational changes of the acyl chains. On the basis of the data from both techniques, we propose possible temperature T–cholesterol composition Xch phase diagrams for these binary bilayers. A notable feature of our phase diagrams, including our previous results for diheptadecanoylphosphatidylcholine (C17PC) and distearoylphosphatidylcholine (C18PC), is that there is a peritectic-like point around Xch=0.15, which can be interpreted as indicating the formation of a 1:6-complex of cholesterol and CnPCs within the binary bilayer irrespective of the acyl chain length. We could give a reasonable explanation for such complex formation using the modified superlattice view. Our results also showed that the Xch value of the abolition of the main transition is almost constant for n=14–17 (ca. 0.33), while it increases to ca. 0.50 for n=18. By contrast, a biphasic n-dependence of Xch was observed for the abolition of the pretransition, suggesting that there are at least two antagonistic n-dependent factors. We speculate that this could be explained by the enhancement of the van der Waals interaction with increases in n and the weakening of the repulsion between the neighboring headgroups with decreases in n.

Cholesteryl Phosphocholine forms Fluid Bilayer Membranes with Saturated or Monounsaturated Ceramides

Some ceramides have been shown to displace cholesterol from liquid ordered domains rich in sphingomyelin or saturated phosphatidylcholine in model membranes. The addition of ceramide will stabilize the formed ceramide/sphingomyelin domains and the subsequent partitioning of cholesterol to the disordered phase will also increase order and stabilization in this phase. The displacement of cholesterol may be due to more favorable interactions of ceramide with the ordered phase lipids, mainly sphingomyelin, compared to cholesterol. The ceramide could also interact less favorably with the disordered phase lipids compared to cholesterol, driving it into the ordered domains. It would also be of interest to better understand how cholesterol and ceramide can interact with each other in bilayer membranes. To study these questions, we have prepared a cholesterol analog with a phosphocholine head group on the 3-oxygen. We have studied the distribution and behavior of chol-PC and ceramides in bilayer systems on their own, and together with saturated and monounsaturated phospholipids. Chol-PC together with ceramide was able to form stable bilayer phases (extruded LUVs, electroformed GUVs). Phosphatidylcholine acyl chain order measurements showed that chol-PC was less efficient in inducing acyl chain order compared to cholesterol. Fluorescence quenching measurements in POPC/ceramide/sterol mixtures further showed a decrease in ordered domain stability of saturated ceramide in the presence of cholesterol or chol-PC. We have also examined the possibility to use chol-PC:ceramide mixtures to load ceramide into cultured cells. Proliferation experiments on HeLa and FRTL-5 cells indicates that chol-PC:ceramide bilayer mixtures are more efficient in inducing apoptosis than DMSO:ceramide mixtures. Our results suggest that chol-PC and ceramide interact with each other under specific conditions, and that the binary bilayers could constitute an interesting formulation for providing cells and tissues with solvent-free ceramides.

Construction of NaREF4-based binary and bilayer nanocrystal assemblies

Monodispersed nanocrystals (NCs) can form ordered and functional binary superlattices. A variety of functional binary nanocrystal superlattices (BNSLs) and bilayer assemblies based on lanthanide-doped NaREF4 (RE = Y, Gd) were constructed with different assembly methods. These assemblies are attractive for applications in photonics as they display multicolor upconversion (UC) emissions.

Improved Coarse-Grained Modeling of Cholesterol Activation in Lipid Bilayers

Cholesterol (Chol) concentration is highly regulated in mammalian cells, which in turn is coupled to LDL levels in blood plasma and cardiovascular health. In one regulatory mechanism, Chol exchange between the plasma and endoplasmic reticulum membranes is regulated by sterol- and sterol derivative-induced changes in membrane structure. Experiments and united atom (UA) simulations show that binary Chol-phospholipid bilayers thin significantly, and Chol accessibility increases, at Chol concentrations of 40 % and higher, which is about the level at which Chol becomes accessible to external acceptors. Here, to reduce computational cost so that larger and more complex systems involved in Chol regulation (i.e. with proteins, ER membranes) can be simulated, we modify the published MARTINI coarse-grained force field to improve its predictions of sterol-induced membrane restructuring, by comparison to united atom simulations. We adjust key bonded parameters and forcefield terms for phospholipid and cholesterol to more accurately reflect the conformational distributions observed in UA simulations, and we further adjust the interaction energies among lipid and sterol beads to reproduce membraneradial distribution functions and the Chol tilt distribution. With these corrections, we are able to reproduce both the area condensation/membrane thickening seen at less than 30% Chol and the thinning seen at above 40%. Furthermore, a “coarse-grained oxysterol” variant of our refined model captures the broad tilt distribution previously seen in UA simulations of bilayers containing 10-18% 25-hydroxycholesterol (oxysterol). Finally, addition of coarse-grained oxysterol to Chol/phospholipid bilayer simulations distinctly shifts the membrane thickness curve to lower concentrations, as seen in experiments. Thus, we have substantially improved the accuracy of MARTINI so that insights can be learned about lipid/sterol systems themselves, and so that large and complex systems also containing proteins can be investigated in the future.

Characterization of the ordered phase formed by sphingomyelin analogues and cholesterol binary mixtures.

The influences of structural alterations of sphingomyelin (SM) on its interactions with cholesterol (chol) and on ordered phase formation were examined by density measurements and surface pressure vs. molecular area isotherm measurements. In addition, we quantitatively characterized the ordered phase formed in each SM and chol binary mixture on the basis of the molecular compressional modulus of SM ( ). Density measurements demonstrated that the ordered phase formation in threo-SM (tSM)/chol and dihydrosphingomyelin (DHSM)/chol binary bilayers shows similar chol concentration-dependency to that of natural erythro-SM (eSM)/chol bilayers; the ordered phase formation was completed in the presence of 25 mol% chol. In contrast, SM bearing a triple bond in the place of a double bond (tripleSM) required a greater concentration of chol to completely transform the bilayer into the ordered phase (at 40 mol% chol). Surface pressure vs. molecular area isotherms showed that the DHSM molecule ( = 290 mN/m) is more rigid than eSM ( = 240 mN/m) above 30 mol% chol (in the ordered phase), although these values are similar (140–150 mN/m) in the absence of chol (liquid condensed phase). Most likely, the DHSM/chol mixture forms a more ordered membrane than the eSM/chol mixture does. Moreover, in the absence of chol, the rigidity of the tripleSM molecule ( = 250 mN/m) is significantly higher as compared with that of the eSM molecule ( = 150 mN/m), which is probably due to the presence of a triple bond.

Effect of the interfacial tension and ionic strength on the thermodynamic barrier associated to the benzocaine insertion into a cell membrane

The insertion of local anaesthetics into a cell membrane is a key aspect for explaining their activity at a molecular level. It has been described how the potency and response time of local anaesthetics is improved (for clinical applications) when they are dissolved in a solution of sodium bicarbonate. With the aim of gaining insight into the physico-chemical principles that govern the action mechanism of these drugs at a molecular level, simulations of benzocaine in binary lipid bilayers formed by DPPC/DPPS were carried out for different ionic strengths of the aqueous solution. From these molecular dynamic simulations, we observed how the thermodynamic barrier associated with benzocaine insertion into the lipid bilayers diminished exponentially as the fraction of DPPS in the bilayer increased, especially when the ionic strength of the aqueous solution increased. In line with these results, we also observed how this thermodynamic barrier diminished exponentially with the phospholipid/water interfacial tension.

Thermotropic Phase Behavior of Binary Bilayer Membrane of Dipalmitoylphosphatidylcholine and Ergosterol

We investigated the phase behavior of a binary bilayer membrane of dipalmitoylphosphatidylcholine (DPPC) and ergosterol, using Prodan fluorescence spectroscopy and differential scanning calorimetry to construct a temperature–composition phase diagram. We found that several important features of the phase diagram can be explained by the superlattice view, on the basis of the hexagonal lattice, which suggests the presence of a molecular cluster composed of an ergosterol molecule and its six surrounding DPPC molecules above an ergosterol composition of ca. 0.15.

A Comparative Study of the Compaction Properties of Binary and Bilayer Tablets of Direct Compression Excipients

Purpose: To comparatively evaluate the tableting properties of binary mixtures and bilayer tablets containing plastic deformation and brittle fracture excipients.Methods: Binary mixture and bilayer tablets of microcrystalline cellulose (MCC), ethyl cellulose, anhydrous lactose and dextrate were prepared by direct compression and the effect of compaction pressure on the materials was investigated by scanning electron microscopy (SEM). True, bulk and tap densities of excipients were determined. Furthermore, Heckel equation and Carr’s index were used to analyze the compression behaviour of the tablets.Results: The flowability of dextrate, based on Heckel and Carr’s Index data, was superior to that of other powder excipients tested. No significant difference was observed between the tensile strength of binary and bilayer tablets of the same composition. However, the tensile strength of binary and bilayer tablets of different compositions varied significantly (p < 0.001), e.g., the tensile strength of microcrystalline cellulose (MCC)/ethyl cellulose (EC) tablets (50/50) was 1.77 MPa while that of MCC/dextrate at 50/50 composition was 1.47 MPa.Conclusion: Binary mixture and bilayer tablets show similar behaviour when formulated using excipients of similar deformation properties. However, their behavior changes when excipients with different deformation properties are blended together.Keywords: Binary mixture, Bilayer tablet, Brittle fracture, Plastic deformation, Tensile strength.

Calorimetric Behavior of Phosphatidylcholine/Phosphatidylethanolamine Bilayers is Compatible with the Superlattice Model

Differential scanning calorimetry was used to study the phase behavior of binary lipid bilayers consisting of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying acyl chain length. A two-state transition model was used to resolve the individual transition components, and the two-state transition enthalpy, the relative enthalpy, and the transition temperature of each component were plotted as a function of composition. Intriguingly, abrupt changes in these thermodynamic parameters were observed at or close to many "critical" X(PE) values predicted by the superlattice model proposing that phospholipids with different headgroups tend to adopt regular rather than random lateral distributions. Statistical analysis indicated that the agreement between the observed and predicted "critical" compositions is highly significant. Accordingly, these data provide strong evidence that the molecules in PC/PE bilayers tend to adopt regular, superlattice-like lateral arrangements, which could be involved in the regulation of the lipid compositions of biological membranes.

Mechanical properties of binary DPPC/DPPS bilayers

In this work, we studied how the lipid composition and ionic strength of an aqueous solution affect the mechanical properties of the lipid bilayer. The interfacial tension, the bending modulus, the Gaussian curvature modulus and the bilayer curvature energy of the lipid bilayer were studied by molecular dynamics simulation. For this purpose, the lipid bilayer was modelled as a binary symmetric lipid bilayer of DPPC (DiPalmitoylPhosphatidylCholine) and DPPS (DiPalmitoylPhosphatidylSerine) at different molar ratios of these two lipids in the absence of salt and in presence of 0.5 N NaCl in aqueous solution. The results of the simulations in absence of salt showed how an increase in the DPPS concentration of the lipid bilayer strongly affects most of its mechanical properties, including the lateral pressure across the membrane, interfacial tension, or the bending modulus of the lipid bilayer. However, in the presence of 0.5 N NaCl, the interfacial tension of the lipid bilayer becomes independent of the lipid composition (in the range of 15–70% DPPS), behavior that may have notable implications from a biological point of view, due to the contribution that this property may provide to the stability of biological membranes.

Interaction of 3β-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes.

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.

Membrane properties of and cholesterol's interactions with a biologically relevant three-chain sphingomyelin: 3O-palmitoyl-N-palmitoyl-D-erythro-sphingomyelin

Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5°C (ΔH 8kcal/mol), which is about 15°C higher than the phase transition temperature of PSM. The 3O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol.

Kinetics for the subgel phase formation in DPPC/DOPC mixed bilayers

We analyzed the kinetics for the subgel (SGI) phase formation in DPPC/DOPC binary bilayers paying attention to DOPC-induced modification of the bilayer physical properties. Differential scanning calorimetry and X-ray diffraction revealed that addition of DOPC reduced the apparent initial lag time to start the SGI phase formation, and that the SGI phase in the binary bilayers had basically the same structure as that in pure DPPC bilayers though addition of DOPC markedly increased the peak temperature and enthalpy of the subtransition in heating. Moreover, addition of DOPC abolished the prolongation of the initial lag time in pure DPPC bilayers induced by lowering the incubation temperature from 0 to −5 °C. Our results suggested that DOPC molecules work as a diffusion enhancer to promote the nucleation of the SGI phase, and relatively destabilize the gel phase so that the formed SGI phase transforms into the ripple phase in heating.

T0201-1-2 せん断応力下での細胞膜構造変化 : 分子シミュレーション([T0201-1]細胞の構造と流れにおけるマイクロ・ナノスケール解析(1))

Structural changes of the cell membrane under shear stresses are investigated by means of molecular dynamics simulations. The cell membrane is modeled as a binary bilayer composed of palmitoyloleoylphosphatidylcholine (POPC) and cholesterol molecules, where the cholesterol concentration is similar to red blood cell's membrane (〜40%). The area per lipid of the binary bilayer is smaller than a pure POPC bilayer, indicating the hydrophobic chains of the lipid become ordered with the inclusion of cholesterol. Shear stresses are imposed by the momentum change of a slab of water molecules adjacent to one side of the bilayer. We found that under shear stresses, the inclusion of cholesterol molecules impairs the deformation of POPC molecules in the bilayer normal direction, whereas it enhances the tilt of POPC molecules.

3P219 Structure and thermal properties of binary lipid bilayers of partially fluorinated and normal phosphatidylcholine(Biol & Artifi memb.: Structure & Property,The 48th Annual Meeting of the Biophysical Society of Japan)

3P219 Structure and thermal properties of binary lipid bilayers of partially fluorinated and normal phosphatidylcholine(Biol & Artifi memb.: Structure & Property,The 48th Annual Meeting of the Biophysical Society of Japan)

The Phase Behavior of Supported Lipid Bilayer Mixtures and Cell Membranes Imaged By Secondary Ion Mass Spectrometry

Supported lipid bilayers have been used as a tool to study the biophysical properties of model membranes with defined compositions. Special attention has been given to the role of cholesterol on the phase behavior of lipid membranes, in particular, to the formation of lipid rafts and complexes. Common techniques used to elucidate the phase behavior of binary and ternary lipid bilayers (i.e. fluorescence and atomic force microscopy) have been limited by their inability to provide direct information on the spatial composition of membranes. Secondary ion mass spectrometry (NanoSIMS) has proved to be a powerful tool for imaging the lateral organization of lipid bilayers with a spatial resolution in the order of tens of nanometers. In this study, a NanoSIMS is used to image lipid bilayers containing isotopically-labeled cholesterol. Additionally, human cell membranes are also imaged.

Nanoscale structure of lipid domain boundaries

Analysis of membrane domain segregation and phase separation is important for the understanding of cell membrane structure and function. In this paper, we report an atomic force microscopy approach using both derivatised tip functionalities and force–volume imaging for the analysis of membrane phase separation. Simultaneous topology and mapping of interaction forces of binary component phospholipid bilayer membranes were performed. Measurements enabled the lateral mapping of bilayer lipid composition at a resolution of 10–20 nm, providing insights into dynamic membrane structure and behaviour. Very sharp boundaries between the two domains are observed in addition to domain boundaries that are quite diffuse with both lipids intermixing. Additionally, some nanoscale domains of one lipid within the domain of the second lipid are observed. The sharp boundaries of the large domains are observed in regions close to these nanodomains.

Highly Stable Poly(Lipid) Bilayers for Long-Term Ion Channel Recordings

Long-term ion channel (IC) screening using cell-based assays is currently limited by throughput and cell to cell variability. ICs isolated and reconstituted into suspended lipid membranes offer an isolated view into IC structure and function, but IC recordings are limited by the short lifetime of the bilayer. Polymerizable lipids (poly(lipids)) offers one potential strategy for long-term investigation and screening of ICs and has been investigated with several model ion channels.The electrical properties and stability of lipid bilayers suspended across glass micropipettes compared natural and polymerized lipids. Breakdown voltage, capacitance, and conductance of the several pure and mixed polymerizable/non-polymerizable lipid bilayers were determined using a patch clamp apparatus.Using, polymerizable phospholipids, we have synthesized membranes with markedly enhanced lifetimes from ca. 3 hours to upwards of 3 weeks. These poly(lipid) bilayers have been used to monitor IC activity of alpha-hemolysin for ca. 1week before loss of alpha-hemolysin. However, poly(lipid) bilayers are rigid and do not support the function of ICs that require membrane fluidity, To address this limitation, binary bilayers composed of poly(lipids) and non-polymerizable lipids were investigated. The resulting mixed bilayers demonstrate markedly enhanced long term stability compared to non-polymerized bilayers and facility IC studies. Alamethicin, a model for voltage-gated ion channels, was shown to be non-functional when reconstituted into homogeneous poly(lipid) bilayers, whereas reconstitution in to mixed bilayers revealed alamethicin activity that and enhanced membrane stability.A functional, truncated form of the KATP channel complex, 6xHis-EGFP-Kir6.2d26, was chosen as a model ligand-gated IC, expressed and purified from yeast. Long-term goals are to reconstitute the truncated version of KATP channels into polymerizable and non-polymerizable bilayers using varying strategies into biomimetric sensing platforms screening ligands and drug candidates for activity.

Imaging cerebroside-rich domains for phase and shape characterization in binary and ternary mixtures

The objective of this paper is to review phase behavior and shape characterization of cerebroside-rich domains in binary and ternary lipid bilayers, as obtained by microscopy techniques. These lipid mixtures provide a format to examine molecular (e.g. headgroup, tail unsaturation, and tail hydroxylation) and thermodynamic (e.g. temperature and mole percentages) factors that determine phase behavior, molecular partitioning, crystal/atomic scale structure, and microstructure/shape (particularly of phase-separated domains). Microscopy can provide excellent spatial (often with high resolution) characterization of cerebroside-rich domains (and their surroundings) to identify, describe or infer with high certainty these characteristics. In the introduction to this review we review briefly the molecular structure, phase behavior, and intermolecular interactions of cerebrosides, in comparison to ceramides and sphingomyelins and in some binary and biological systems. The bulk of the review is then devoted to microscopy investigations of cerebroside-rich domain microstructure and shape dynamics in binary and ternary (one component is cholesterol) systems. Quantitative and/or high-resolution microscopy techniques have been used to interrogate cerebroside-rich domains such as freeze-fracture electron microscopy, atomic force microscopy, imaging elipsometry, two-photon fluorescence microscopy, and LAURDAN generalized polarization in addition to the laboratory workhorse technique of epifluorescence microscopy that allows a quick often qualitative assessment of microstructure and dynamics. We particularly focus on the information these microscopy investigations have revealed with respect to phase behavior, cholesterol partitioning, domain shape, and determinants of domain shape.