Bilayer Mixtures Research Articles

Cholesterol drives enantiospecific effects of ibuprofen in biomimetic membranes

The interaction between chiral drugs and biomimetic membranes is of interest in biophysical research and biotechnological applications. There is a belief that the membrane composition, particularly the presence of cholesterol, could play a pivotal role in determining enantiospecific effects of pharmaceuticals. Our study explores this topic focusing on the interaction of ibuprofen enantiomers (S- and R-IBP) with cholesterol-containing model membranes. The effects of S- and R-IBP at 20 mol% on bilayer mixtures of dipalmitoylphosphatidylcholine (DPPC) with 0, 10, 20 and 50 mol% cholesterol were investigated using circular dichroism and spin-label electron spin resonance. Morphological changes due to IBP enantiomers were studied with atomic force microscopy on supported cholesterol-containing DPPC monolayers. The results reveal that IBP isoforms significantly and equally interact with pure DPPC lipid assemblies. Cholesterol content, besides modifying the structure and the morphology of the membranes, triggers the drug enantioselectivity at 10 and 20 mol%, with the enantiomers differently adsorbing on membranes and perturbing them. The spectroscopic and the microscopic data indicate that IBP stereospecificity is markedly reduced at equimolar content of Chol mixed with DPPC. This study provides new insights into the role of cholesterol in modulating enantiospecific effects of IBP in lipid membranes.

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

OxPAPC stabilizes liquid-ordered domains in biomimetic membranes

Long-chain polyunsaturated fatty acids (PUFAs) are prone to nonenzymatic oxidation in response to differing environmental stressors and endogenous cellular sources. There is increasing evidence that phospholipids containing oxidized PUFA acyl chains control the inflammatory response. However, the underlying mechanism(s) of action by which oxidized PUFAs exert their functional effects remain unclear. Herein, we tested the hypothesis that replacement of 1-palmitoyl-2-arachidonyl-phosphatidylcholine (PAPC) with oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC) regulates membrane architecture. Specifically, with solid-state 2H NMR of biomimetic membranes, we investigated how substituting oxPAPC for PAPC modulates the molecular organization of liquid-ordered (Lo) domains. 2H NMR spectra for bilayer mixtures of 1,2-dipalmitoylphosphatidylcholine-d62 (an analog of DPPC deuterated throughout sn-1 and -2 chains) and cholesterol to which PAPC or oxPAPC was added revealed that replacing PAPC with oxPAPC disrupted molecular organization, indicating that oxPAPC does not mix favorably in a tightly packed Lo phase. Furthermore, unlike PAPC, adding oxPAPC stabilized 1,2-dipalmitoylphosphatidylcholine-d6-rich/cholesterol-rich Lo domains formed in mixtures with 1,2-dioleoylphosphatidylcholine while decreasing the molecular order within 1,2-dioleoylphosphatidylcholine-rich liquid-disordered regions of the membrane. Collectively, these results suggest a mechanism in which oxPAPC stabilizes Lo domains—by disordering the surrounding liquid-disordered region. Changes in the structure, and thereby functionality, of Lo domains may underly regulation of plasma membrane-based inflammatory signaling by oxPAPC.

Identification of possible binding modes of SARS-CoV-2 spike N-terminal domain for ganglioside GM1

Coarse-grained molecular dynamics simulations of the lipid bilayer mixture of POPC and cholesterol were carried out in the presence and absence of ganglioside monosialo 1 (GM1) with N - terminal domain (NTD) of SARS-CoV-2 spike glycoprotein. The interactions of GM1 with two different NTD orientations were compared. NTD orientation I compactly bind GM1 predominantly through the sialic acid and the external galactose moieties providing more restriction to GM1 mobility whereas orientation II is more distributed on the lipid surface and due to the relaxed mobility of GM1 there, presumably, the NTD receptor penetrates more through the membrane.

Molecular mechanisms of spontaneous curvature and softening in complex lipid bilayer mixtures

Membrane reshaping is an essential biological process. The chemical composition of lipid membranes determines their mechanical properties and thus the energetics of their shape. Hundreds of distinct lipid species make up native bilayers, and this diversity complicates efforts to uncover what compositional factors drive membrane stability in cells. Simplifying assumptions, therefore, are used to generate quantitative predictions of bilayer dynamics based on lipid composition. One assumption commonly used is that “per lipid” mechanical properties are both additive and constant—that they are an intrinsic property of lipids independent of the surrounding composition. Related to this is the assumption that lipid bulkiness, or “shape,” determines its curvature preference, independently of context. In this study, all-atom molecular dynamics simulations on three separate multilipid systems were used to explicitly test these assumptions, applying methodology recently developed to isolate properties of single lipids or nanometer-scale patches of lipids. The curvature preference experienced by populations of lipid conformations were inferred from their redistribution on a dynamically fluctuating bilayer. Representative populations were extracted by both structural similarity and semi-automated hidden Markov model analysis. The curvature preferences of lipid dimers were then determined and compared with an additive model that combines the monomer curvature preference of both the individual lipids. In all three systems, we identified conformational subpopulations of lipid dimers that showed non-additive curvature preference, in each case mediated by a special chemical interaction (e.g., hydrogen bonding). Our study highlights the importance of specific chemical interactions between lipids in multicomponent bilayers and the impact of interactions on bilayer stiffness. We identify two mechanisms of bilayer softening: diffusional softening, driven by the dynamic coupling between lipid distributions and membrane undulations, and conformational softening, driven by the inter-conversion between distinct dimeric conformations.

Highly Selective Supported Graphene Oxide Membranes for Water-Ethanol Separation

A polyethersulfone (PES)-supported graphene oxide (GO) membrane has been developed by a simple casting approach. This stable membrane is applied for ethanol/water separation at different temperatures. The 5.0 µm thick GO film coated on PES support membrane showed a long-term stability over a testing period of one month and excellent water/ethanol selectivity at elevated temperatures. The water/ethanol selectivity is dependent on ethanol weight percentage in water/ethanol feed mixtures and on operating temperature. The water/ethanol selectivity was enhanced with an increase of ethanol weight percentage in water/ethanol mixtures, from below 100 at RT to close to 874 at a 90 °C for 90% ethanol/10% water mixture. Molecular dynamics simulation of water-ethanol mixtures in graphene bilayers, that are considered to play a key role in transport, revealed that molecular transport is negligible for layer spacing below 1 nm. The differences in the diffusion of ethanol and water in the bilayer are not consistent with the large selectivity value experimentally observed. The entry of water and ethanol into the interlayer space may be the crucial step controlling the selectivity.

Microscopic Insights into Biological Relevance of Membrane Channels in Gas Transport across Lipid Membranes

Membrane transport of small, non-polar gas molecules, such as O2 and CO2, is among fundamental processes of living cells. Because of their size and chemical properties, these molecules may unimpededly transit through the lipid phase of the membrane. However, varying degrees of gas permeability in membranes with different lipid compositions have been reported experimentally, suggesting involvement of protein channels, particularly aquaporins (AQPs), in gas transport across membranes. To account for membrane heterogeneity and to probe potential roles of AQPs in gas transport, we performed extensive MD simulations of gas diffusion through lipid membranes. The systems simulated included POPC bilayers embedded with an AQP tetramer (i.e., AQP1, AQP5 or AQP7), and protein-free lipid bilayer mixtures of POPC, cholesterol (CHL) and sphingomyelin (SM) or DPPC. Each of them contained an excess amount (125 molecules) of a gas species and was simulated for several hundred nanoseconds. Since the gas molecules were allowed to diffuse freely, their permeability coefficients were directly estimated by measuring their flow through the lipid and protein phases. The results showed that gas molecules diffused through the membrane via monomeric water and hydrophobic central pores of AQPs, and via the lipid phase. For fluid-like membranes (e.g., 100% POPC) which exhibited high gas permeability, AQPs would not facilitate the permeation. Highly-ordered or gel-like membranes (e.g., 100% SM and 50:50 SM:CHL or DPPC:CHL), on the other hand, exhibited reduced gas permeability as low as or even lower than through AQPs, suggesting that in such conditions, AQPs would become imminent in facilitating gas transport. Consistently with experimentally observed low gas permeability in high SM-CHL containing membranes, such as those of erythrocytes and ocular lens, our study suggests biological significance of protein-facilitated gas permeation across membranes.

Characterization of Interactions between Curcumin and Different Types of Lipid Bilayers by Molecular Dynamics Simulation.

Curcumin (CUR) is a natural food ingredient with known ability to target microbial cell membrane. In this study, the interactions of CUR with different types of model lipid bilayers (POPE, POPG, POPC, DOPC, and DPPE), mixtures of model lipid bilayers (POPE/POPG), and biological membrane mimics (Escherichia coli and yeast) were investigated by all-atom explicit solvent molecular dynamics (MD) simulation. CUR readily inserts into different types of model lipid bilayer systems in the liquid crystalline state, staying in the lipid tails region near the interface of lipid head and lipid tail. Parallel orientation to the membrane surface is found to be more probable than perpendicular for CUR, as indicated by the tilt angle distribution. This orientation preference is less significant as the fraction of POPE is increased in the system, likely due to the better water solvation of perpendicular orientation in the POPE bilayer. In E. coli and yeast bilayers, tilt angle distributions were similar to that for POPE/POPG mixed bilayer, with water hydration number around CUR for the former being higher. Insertion of CUR resulted in membrane thinning. The results from these simulations provide insights into the possible differences in membrane disrupting activity of CUR against different types of microorganisms.

Nanoscale Structure of Lipid Bilayers Revealed By In-Silico and Experimental Small Angle Neutron Scattering

Through the combination of simulations and numerical analysis with small angle neutron scattering, we probe the lateral organization of lipid bilayer mixtures. Small Angle Neutron Scattering (SANS) is an excellent complement to optical techniques, as it reveals molecular scale structural details without fluorescent probes. On its own, however, SANS is not sufficient to determine the organization of the membrane. We therefore take a two-pronged approach, comparing molecular dynamics simulations of ternary mixtures to experimental data obtained for the same mixtures. In-silico neutron scattering experiments on simulated membranes are compared to the experimental data, simultaneously validating the simulation data and providing a molecular scale model of the experimental system. Our all-atom molecular dynamics simulations use pre-existing software packages (e.g., Gromacs) to produce atomic trajectories of ten microseconds in length. These trajectories are processed and analyzed using custom software to generate scattering intensities for comparison to experiment, assuming laterally averaged scattering within nanoscopic domains. Simulated scattering intensities are fit to experimental data to extract information about the lateral structure. In this way, the transverse variation in scattering length density across the bilayer — which, under fully deuterated solvent, is much more significant than lateral contributions — is used to infer lateral structure.

Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin.

Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data.

Line Tension Controls Liquid-Disordered + Liquid-Ordered Domain Size Transition in Lipid Bilayers

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.

Molecular dynamics simulation of cytotoxicity of graphene nanosheets to blood-coagulation protein.

Graphene is a nanomaterial that is widely used in electronics, biomedicine, and drug-delivery systems. Although it has many industrial applications, the cytotoxicity of graphene has not been sufficiently studied. In this study, the authors used molecular dynamics simulation to investigate how a graphene nanosheet affects a blood-coagulation protein, namely, a tissue factor/FVIIa binary complex bound to a lipid bilayer membrane, in a 4:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine lipid bilayer mixture. Based on the results, the authors suggest a mechanism for the cytotoxicity of graphene nanosheets to blood-coagulation protein at the molecular level.

Effects of Ester and Ether Linkage in Phospholipids on Ld + Lo Domain Size Transition for a Four-Component Lipid Bilayer Mixture

Effects of Ester and Ether Linkage in Phospholipids on Ld + Lo Domain Size Transition for a Four-Component Lipid Bilayer Mixture

Bilayer Deformation, Pores, and Micellation Induced by Oxidized Lipids.

The influence of different oxidized lipids on lipid bilayers was investigated with 16 individual 1 μs atomistic molecular dynamics (MD) simulations. Binary mixtures of lipid bilayers of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) and its peroxide and aldehyde products were performed at different concentrations. In addition, an asymmetrical short chain lipid, 1-palmitoyl-2-decanoyl-sn-glycero-3-phosphatidylcholine (PDPC), was used to compare the effects of polar/apolar groups in the lipid tail on lipid bilayer. Although water defects occurred with both aldehyde and peroxide lipids, full pore formation was observed only for aldehyde lipids. At medium concentrations the pores were stable. At higher concentrations, however, the pores became unstable and micellation occurred. Data analysis shows that aldehyde lipids' propensity for pore formation is due to their shorter and highly mobile tail. The highly polar peroxide lipids are stabilized by strong hydrogen bonds with interfacial water.

Macroscopic and Nanoscopic Heterogeneous Structures in a Three-Component Lipid Bilayer Mixtures Determined by Atomic Force Microscopy.

Much of lipid raft properties can be inferred from phase behavior of multicomponent lipid membranes. We use liquid compatible atomic force microscopy (AFM) to study a three-component system composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (eSM), and cholesterol. Specifically, we obtain macroscopic and nanoscopic heterogeneous structures in a broad compositional space of DOPC/eSM/cholesterol (23 °C). In the macroscopic liquid coexisting region, we quantify area fraction of the coexisting phases and determine a set of thermodynamic tie-lines. When lipid compositions are near the critical point, we obtain fluctuation-like nanoscopic structures. We also use AFM height images to explore the hypothetical three-phase coexisting region. Finally, we use fluorescence microscopy to compare the phase behavior from our AFM measurements to that in free-floating giant unilamellar vesicles (GUVs). Our results highlight the role of lipid composition in mediating lipid domain formation and stability.

Appearance of Modulated Bilayer Morphology for Coexisting LD and LO Phases is Correlated with Line Tension

Giant Unilamelar Vesicles (GUVs) appear uniform for the brain-SM/POPC/Chol bilayer mixture, whereas GUVs exhibit macroscopic domains of coexisting liquid-disordered + liquid-ordered phases for the brain-SM/DOPC/Chol mixture. We can travel through composition space in order to study this transition from nanodomains with POPC to macrodomains with DOPC. Using ρ defined as the ratio [DOPC]/[DOPC]+[POPC], the domain morphology undergoes a transition regime where “modulated phases” appear as a function of ρ. The formation of these different morphologies on a GUV can be understood as a competition between line tension, which favors large domains, and bending energy, which favors small domains (Amazon et al). We measured the ρ values where modulated phases appear for different 4-component mixtures, using brain-SM, or egg-SM, or palmitoyl-SM as the high-melting lipid, and DOPC/POPC or DOPC/SOPC as the low melting lipids, for a total of six different 4-component mixtures. We then measured the line tension of the macroscopic domains vs ρ, and found the same line tension at ρ values at the transition between modulated phases and macroscopic phases. This finding implies that line tension has major control over domain morphology. We are currently studying how the transmembrane peptide, GWALP-23, changes the morphology by changing the values of the competing interactions.

Acceleration of Lateral Equilibration in Mixed Lipid Bilayers Using Replica Exchange with Solute Tempering.

The lateral heterogeneity of cellular membranes plays an important role in many biological functions such as signaling and regulating membrane proteins. This heterogeneity can result from preferential interactions between membrane components or interactions with membrane proteins. One major difficulty in molecular dynamics simulations aimed at studying the membrane heterogeneity is that lipids diffuse slowly and collectively in bilayers, and therefore, it is difficult to reach equilibrium in lateral organization in bilayer mixtures. Here, we propose the use of the replica exchange with solute tempering (REST) approach to accelerate lateral relaxation in heterogeneous bilayers. REST is based on the replica exchange method but tempers only the solute, leaving the temperature of the solvent fixed. Since the number of replicas in REST scales approximately only with the degrees of freedom in the solute, REST enables us to enhance the configuration sampling of lipid bilayers with fewer replicas, in comparison with the temperature replica exchange molecular dynamics simulation (T-REMD) where the number of replicas scales with the degrees of freedom of the entire system. We apply the REST method to a cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer mixture and find that the lateral distribution functions of all molecular pair types converge much faster than in the standard MD simulation. The relative diffusion rate between molecules in REST is, on average, an order of magnitude faster than in the standard MD simulation. Although REST was initially proposed to study protein folding and its efficiency in protein folding is still under debate, we find a unique application of REST to accelerate lateral equilibration in mixed lipid membranes and suggest a promising way to probe membrane lateral heterogeneity through molecular dynamics simulation.

Partitioning of lysolipids, fatty acids and their mixtures in aqueous lipid bilayers: Solute concentration/composition effects

Distributions of lysopalmitoylphosphatidylcholine (LPPC), palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects only the solutes in water. Water solute concentrations were obtained at each of several lipid concentrations. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in the concentration range investigated. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the free energy of transfer, for each of these solutes were determined from the fit. Main findings are: (1) Water/bilayer partition coefficient of solute is greater for 2 to 10% solute mole fraction than for 0 to 2%, signaling solute induced bilayer perturbation that increases bilayer solubility, beginning at 2% solute mole fraction. (2) Partition coefficients are in the order LPPC<PA<LPPC+PA at 37°C and LPPC+PA≤LPPC<PA at 50°C. This signifies synergism toward increased solute solubility in the bilayer-gel phase and lack of it in the bilayer-liquid phase when LPPC and PA are present together. Implications of the solute concentration/composition and bilayer phase dependences of the partition coefficients to the reported solute induced enhancements in transmembrane permeability are discussed.

Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol

We report the first 4-component phase diagram for the lipid bilayer mixture, DSPC/DOPC/POPC/chol (distearoylphosphatidylcholine/dioleoylphosphatidylcholine/1-palmitoyl, 2-oleoylphosphatidylcholine/cholesterol). This phase diagram, which has macroscopic Ld+Lo phase domains, clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has nanoscopic phase domains of Ld+Lo. Our studies start from two published ternary phase diagrams, and show how these can be combined into a quaternary phase diagram by study of a few hundred samples of intermediate compositions.

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.