Mixed Bilayers Research Articles

Pinning of domains for fluid–fluid phase separation in lipid bilayers with asymmetric dynamics

We propose a physical mechanism for the arrest of domain coarsening in a system of two apposed two-dimensional binary fluids. The two fluids are subject to a dynamic asymmetry: strong friction with the environment allows domains in one fluid layer (the “bottom” fluid) to grow only diffusively, whereas hydrodynamic flow leads to initially faster growth in the apposed fluid (the “top” layer). The two fluids are energetically coupled so that domains of similar type interact favorably across the two fluids. Using lattice Boltzmann simulations we observe that at a certain length scale, which is independent of the coarsening state in the bottom layer, domain growth in the top layer comes to an arrest. A phenomenological model suggests the pinning of domains across the two fluids to cause the arrest in domain growth. The pinning results from the interplay between line tension and domain coupling strength across the two fluids. We apply our model to a lipid bilayer for which we calculate the length scale of the dynamically arrested domains in the top layer. We find domain extensions of about or somewhat larger than 20 nm. Potential applications of our pinning model are to mixed lipid bilayers that tend to phase separate and are subject to a dynamic asymmetry; these include model membranes on a solid support and lipid rafts in the plasma membrane.

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

Polyunsaturated lipids are remarkably flexible molecules, with a great influence on the membrane structure and dynamics, affecting from mechanical properties to domain segregation. In this work, we studied phospholipid mixtures of dipalmitoylphosphatidylcholine (DPPC) and diunsaturated phosphatidylcholine lipids (diunsPC) of different lengths, by means of molecular dynamic simulations of a coarse-grained interaction model. These diunsPC:DPPC binary mixtures show nonideal behavior characterized by one mixed phase with composition fluctuations on a length scale of nanometers. Motivated by this observation, we studied comprehensively the characteristics of molecular structure as a function of the compositional gradient. We analyzed orientational order profiles, density distributions, and pair-pair correlation functions between the molecule residues. We observed that, in diunsPC-enriched regions, DPPC tails become expanded and disordered, especially toward the membrane center. On the other hand, in the more condensed DPPC-enriched patches, diunsaturated acyl chains become displaced toward the interface instead of stretching along the membrane normal. From the comparison of the two diunsPC lipids of different tail length, we measured that the presence of a longer terminal saturated segment induces better mixing with DPPC, and most interestingly eliminates the up-down composition correlation measured with the shorter tail-diunsPC. At molecular level, there is a reduced redistribution of densities and changes in the local order as a function of composition. We interpret these results as indicative that the packing incompatibility between polyunsaturated and saturated lipids rules their mixing behavior.

Segregation of Negatively Charged Phospholipids by the Polycationic and Farnesylated Membrane Anchor of Kras

The Kras protein, a member of the Ras family of bio-switches that are frequently mutated in cancer and developmental disorders, becomes functional when anchored to the inner surface of the plasma membrane. It is well known that membrane attachment involves the farnesylated and poylcationic C-terminus of the protein. However, little is known about the structure of the complex and the specific protein-lipid interactions that are responsible for the binding. On the basis of data from extensive (>0.55 μs) molecular dynamics simulations of multiple Kras anchors in bilayers of POPC/POPG lipids (4:1 ratio), we show that, as expected, Kras is tethered to the bilayer surface by specific lysine-POPG salt bridges and by nonspecific farnesyl-phospholipid van der Waals interactions. Unexpectedly, however, only the C-terminal five of the eight Kras Lys side chains were found to directly interact with the bilayer, with the N-terminal ones staying in water. Furthermore, the positively charged Kras anchors pull the negatively charged POPG lipids together, leading to the clustering of the POPG lipids around the proteins. This selective Kras-POPG interaction is directly related to the specific geometry of the backbone, which exists in two major conformational states: 1), a stable native-like ensemble of structures characterized by an extended geometry with a pseudohelical turn; and 2), less stable nonnative ensembles of conformers characterized by severely bent geometries. Finally, although the interface-bound anchor has little effect on the overall structure of the bilayer, it induces local thinning within a persistence length of ∼12 Å. Our results thus go beyond documenting how Kras attaches to a mixed bilayer of charged and neutral lipids; they highlight a fascinating process of protein-induced lipid sorting coupled with the (re)shaping of a surface-bound protein by the host lipids.

Simulating POPC and POPC/POPG Bilayers: Conserved Packing and Altered Surface Reactivity.

Molecular dynamics (MD) simulation is a popular technique to study bilayer structural properties, but it has not been widely used in mixed bilayers of neutral and charged lipids. Here, we present results from constant temperature and pressure MD simulations of a 2-oleoyl-1-pamlitoyl-sn-glyecro-3-phosphocholine (POPC) bilayer containing 23% 2-oleoyl-1-pamlitoyl-sn-glyecro-3-glycerol (POPG). The simulations were performed using the recently updated CHARMM force field and involved two bilayers of 104 and 416 lipids. A control simulation of a pure POPC bilayer of 128 lipids yielded equilibrium structural properties that compare very well with experimental data. The average equilibrium properties of the mixed bilayer systems were very similar to those of the pure POPC. However, nearly one-half of all the POPG lipids were found to be involved in hydrogen bonding with POPC lipids. Furthermore, the hydration of the mixed bilayer is different from that of the pure POPC, with the former inducing ordering of water molecules at longer distances. Thus, a phospholipid bilayer with ∼23% negative charge content in the liquid crystalline phase differs from its neutral counterpart only at the headgroup.

The vesicle-to-micelle transformation of phospholipid–cholate mixed aggregates: A state of the art analysis including membrane curvature effects

We revisited the vesicle-to-micelle transformation in phosphatidylcholine–cholate mixtures paying special attention to the lipid bilayer curvature effects. For this purpose, we prepared unilamellar vesicles with different starting sizes (2rv=45–120nm). We then studied mixtures of the unilamellar vesicles (1–8mmol kg–1) and sodium cholate (0–11.75mmolkg−1) by static and dynamic light scattering. The transformation generally comprises at least two, largely parallel phenomena; one increases and the other decreases the average mixed aggregate size. In our view, cholate first induces bilayer fluctuations that lead to vesicle asphericity, and then to lipid bilayer poration followed by sealing/reformation (or fusion). The cholate-containing mixed bilayers, whether in vesicular or open form, project thread-like protrusions with surfactant enriched ends even before complete bilayer solubilisation. Increasing cholate concentration promotes detachment of such protrusions (i.e. mixed micelles formation), in parallel to further softening/destabilising of mixed amphipat bilayers over a broad range of concentrations. Vesicles ultimately fragment into mixed thread-like micelles. Higher cholate relative concentrations yield shorter thread-like mixed micelles. Most noteworthy, the cholate-induced bilayer fluctuations, the propensity for large aggregate formation, the transformation kinetics, and the cholate concentration ensuring complete lipid solubilisation all depend on the starting mean vesicle size. The smallest tested vesicles (2rv=45nm), with the highest bilayer curvature, require ~30% less cholate for complete solubilisation than the largest tested vesicles (2rv=120nm).

Effects of β-Cyclodextrin on the Structure of Sphingomyelin/Cholesterol Model Membranes

The interaction of β-cyclodextrin (β-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after β-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by β-CD, indicating that β-CD is unable to remove sphingomyelin or complexed Chol. However, β-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by β-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of β-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by β-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane.

Retinoschisin (RS1) Interacts with Negatively Charged Lipid Bilayers in the Presence of Ca2+: An Atomic Force Microscopy Study

Retinoschisin (RS1) is a retina-specific secreted protein encoding a conserved discoidin domain sequence. As an adhesion molecule, RS1 preserves the retinal cell architecture and promotes visual signal transduction. In young males, loss-of-function mutations in the X-linked retinoschisis gene (RS1) cause X-linked retinoschisis, a form of progressive blindness. Neither the structure of RS1 nor the nature of its anchoring and organization on the plasma membranes is fully understood. The discoidin C2 domains of coagulation factors V and VIII are known to interact with extracellular phosphatidylserine (PS). In this study we have used atomic force microscopy (AFM) to study the interactions of murine retinoschisin (Rs1) with supported anionic lipid bilayers in the presence of Ca(2+). The bilayers consisting of a single lipid, PS, and mixtures of lipids with or without PS were used. Consistent with previous X-ray diffraction studies, AFM imaging showed two distinct domains in pure PS bilayers when Ca(2+) was present. Upon Rs1 adsorption, these PS and PS-containing mixed bilayers underwent fast and extensive reorganization. Protein localization was ascertained by immunolabeling. AFM imaging showed the Rs1 antibody bound exclusively to the calcium-rich ordered phase of the bilayers pointing to the sequestration of Rs1 within those domains. This was further supported by the increased mechanical strength of these domains after Rs1 binding. Besides, changes in bilayer thickness suggested that Rs1 was partially embedded into the bilayer. These findings support a model whereby the Rs1 protein binds to PS in the retinal cell plasma membranes in a calcium-dependent manner.

Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na+) on the formation of salt-bridges is investigated. In particular, the number of Na+-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38 degrees C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20 degrees -50 degrees C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

Atomic force microscopy of supported lipid membranes and their complexes with polycations

The method of atomic force microscopy has been used to investigate the morphology of mica-supported bilayer lipid membranes and stability of their complexes with a cationic polymer, poly-(N-ethyl-4-vinylpyridinium bromide). Lipid bilayers with a minimum of defects were obtained by the fusion of monolamellar neutral or mixed anionic bilayer vesicles (liposomes) on the mica surface, followed by excessive solvent removal by means of rapid rotation of a plate in horizontal plane (spin-coating). It has been shown that the cationic polymer does not interact with the bilayers, where the outer leaflet (i.e., the monolayer exposed to the surrounding aqueous solution) is made of an electroneutral phosphatidylcholine (PC). At the same time, the polymer irreversibly binds to the bilayer containing an anionic lipid.

Molecular and component volumes of saturated n-alkanols in DOPC + DOPS bilayers

The volumetric properties of fluid bilayers consisting of dioleoylphosphatidylcholine (DOPC, 96 wt%) and dioleoylphosphatidylserine (DOPS, 4 wt%) with incorporated saturated n-alkanols (CnOH, n = 10–16 is the even number of carbons in alkyl chain) were studied by vibrating tube densitometry. The mixing of DOPC and DOPS was found to be ideal and the molecular volumes of pure lipids V DOPC and V DOPS are additive in mixed bilayers. The increase of V DOPS with temperature was steeper than that of V DOPC as quantified by significantly higher coefficient of isobaric thermal expansivity γ. This difference is supposed to be related to the high thermal expansivity of the serine headgroup. The molecular volumes of lipids and CnOH ( V CnOH) in fluid DOPC + DOPS bilayers are constant and additive within error limits for n = 12 and 16 and for the CnOH mole fractions in the lipid bilayer x AL ≤ 0.5 and x AL ≤ 0.3, respectively. In homologous series of CnOHs incorporated into DOPC + DOPS bilayer, the V CnOH volume increases linearly with the CnOH chain length n at x AL = 0.29. From this dependence obtained at several temperatures, the component volume of the CnOH hydroxyl group V OH was calculated and found to decrease between 20 °C and 51 °C while the component volume of the CnOH methylene group V C H 2 increases with temperature. The excess molecular volume, Δ V, for the transfer of CnOH into the DOPC + DOPS bilayers display a biphasic dependence on the alkyl length, n, with a minimum for n = 12. When calculated with respect to the pure CnOH, Δ V is negative for homologs with n > 8.

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.

Computer Simulations of Alzheimer's Beta Amyloid Interactions with Multicomponent Lipid Bilayers

Amyloidogenic protein unfolding and subsequent aggregation on cell surfaces are linked to many protein misfolding diseases, e.g., Alzheimer's (AD) and Parkinson's. Beta-amyloid (betaA), a 39 to 43 residue peptide, is released from neuronal membranes upon sequential proteolytic cleavages of a large transmembrane amyloid precursor protein by two secretases. An understanding of the conformational transitions and stability mediated by the lipid surface interactions is important for developing new strategies for the prevention and treatment of protein misfolding diseases. Using all-atom MD simulation techniques, we explore the initial folding and lipid insertion kinetics of betaA of both 40 and 42 residue long on the surfaces of well-defined lipid nanodomains with different cholesterol contents that mimic the neuronal lipid membranes. Several molecular clusters consisting of different initial conformations, alpha-barrel, beta-sheet and globular-coil, of betaA and stable mixed lipid bilayer in explicit solvent have successfully been constructed. The conformational transitions and stability of betaA on the lipid surface and in a partially inserted state on lipid nanodomains of different lipid compositions were systematically studied. The protein-induced membrane disruptions were examined by calculating the lipid order parameter, water permeability and bilayer thickness profiles of the lipid bilayers. The time-dependent secondary structure of betaA was used to gauge the unfolding events of the protein and its dependence on the lipid composition of the interacting bilayers. Our results revealed that the cholesterol content in the lipid bilayer strongly affects the initial lipid surface-induced unfolding and bilayer-insertion and stability behavior of betaA in our model bilayer systems. Our computer simulation data may provide useful computational insights on the controversial sensitization and protective roles of lipid membranes on the protein aggregation events in AD.

Flip-Flop Motions of Lipid Molecules in Mixed Bilayer Systems

The cell membrane is composed of a wide variety of lipid molecules, cholesterols, and membrane proteins. Lipid molecules in the membrane have several time scales of motions ranging from femtosecond to seconds. The flip-flop motion, in which lipid molecules move from one leaflet to the other, is known to be one of the slowest: it typically occurs within several tens of seconds, or much longer. Recently, experimental studies revealed that cholesterols (CHOL), diacylglycerols (DAG), and ceramides (CER) show fast flip-flop motions in some membranes. However, the molecular mechanisms underling the motions remain elusive. In this work, we performed coarse-grained molecular dynamics simulations, using MALTINI force field parameters. We examined flip-flop motions of CHOL, DAG, and CER in phospholipid bilayer systems, composing of DAPC(di-20:4), SAPC(18:0-20:4), and POPC(16:0-18:1). In the simulations using DAPC membranes, we observed flip-flop motions of CHOL, DAG, and CER within a microsecond. The flip-flop rate of CHOL was the highest, whereas that of DAG was lower than CHOL. CER flipped only once during the simulation. This tendency of flip-flop motions is strongly correlated with the relative positions of the lipids to the bilayer membranes: CHOL stays almost at the center of the membrane, whereas the head group of CER is located at the water/membrane interface and interacted with solvent molecules strongly. The flip-flop motions of lipids were also affected with the membrane environment. Within 1-microsecond simulations, CHOL flipped 257 times in DAPC, 196 times in SAPC, and 5 times in POPC. Thus, the flip-flop rate is strongly correlated with the number of double bonds in the acyl chains of bilayer phospholipids, suggesting the importance of the membrane fluidity. These simulation results qualitatively agree with existing experimental data and shed light on the molecular mechanisms underlying the dynamics of biomembranes.

3P224 Lipid dynamics in mixed bilayer systems with micro domains of cholesterol and diacylglycerol(Biol & Artifi memb.: Dynamics,The 48th Annual Meeting of the Biophysical Society of Japan)

3P224 Lipid dynamics in mixed bilayer systems with micro domains of cholesterol and diacylglycerol(Biol & Artifi memb.: Dynamics,The 48th Annual Meeting of the Biophysical Society of Japan)

Chemical Evolution of Amphiphiles: Glycerol Monoacyl Derivatives Stabilize Plausible Prebiotic Membranes

The self-assembly of simple amphiphiles like fatty acids into cell-like membranous structures suggests that such structures were available on prebiotic Earth to support the origin of cellular life. However, the composition of primitive membranes remains unclear because the physical properties of the aqueous environment in which they assembled are relatively unconstrained in terms of temperature, pH, and ionic concentrations. It seems likely that early membranes were composed of mixtures of various amphiphiles in an aqueous medium warmed by geothermal activity prevalent in the Archean era. To better understand the properties of mixed bilayers formed by binary mixtures of single-chain amphiphiles under these conditions, we conducted stability experiments, using membranes composed of various fatty acids having hydrocarbon chain length between 8 and 18 carbons, in mixtures with their glycerol monoacyl amphiphile derivatives (GMAs). The parameters investigated were critical vesicle concentration (CVC), encapsulation, and temperature-dependent stability. We found that hydrocarbon chain length and the presence of GMAs were major factors related to membrane stability. As chain length increased, GMA additions decreased the CVC of the mixtures 4- to 9-fold. Encapsulation ability also increased significantly as a function of chain length, which reduced permeation of small marker molecules. However, long exposures to temperatures in excess of 60 degrees C resulted in a total release of encapsulated solutes and extensive mixing of the membrane components between vesicles. We conclude that GMAs can significantly increase the stability of mixed amphiphile membranes, but further studies are required to establish model membranes that are stable at elevated temperatures.

The Location of the Biosurfactant Surfactin in Phospholipid Bilayers Supported on Silica Using Neutron Reflectometry

We have investigated the formation of supported surfactin-phospholipid mixed bilayers using neutron reflectometry. Micellar mixtures of phospholipid (diphosphatidyl choline, DPPC), surfactin, and beta-d-dodecyl maltoside were used to make the deposition. When the surfactin concentration is at its critical micelle concentration (CMC = 6 x 10(-6) M) in the bulk solution, there is no adsorption at all on the silica. When the surfactin concentration is lowered below the CMC, a mixed bilayer of surfactin and DPPC is formed. Since surfactin does not adsorb on silica from solutions of surfactin alone, this shows that there is a strong attraction between surfactin and DPPC. The variation of adsorbed amount, composition, and structure of the adsorbed layer are consistent with the attractive interaction between surfactin and DPPC and with their respective negative and positive affinities for the silica surface. Three phospholipid isotopic contrasts were measured and used to define the composition and structure of the surfactin-phospholipid bilayer. The maximum amount of surfactin in the bilayer reaches a mole fraction of about 0.2 and this is located in the outer leaflet of the bilayer within the headgroup and part of the adjacent chain region.

Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers

The interaction with model membranes of a peptide, EqtII(1-32), corresponding to the N-terminal region of the pore-forming toxin equinatoxin II (EqtII) has been studied using solid-state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [(19)F-para]Phe16-[1-(13)C]Leu19 and [(19)F-para]Phe16-[(15)N]Leu23 analogs of EqtII(1-32) measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16-23, in the presence of bilayers. (19)F-(31)P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII(1-32) in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII(1-32) in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC-bound peptide adopting helical formations at residues 16-24, whereas the DMPC:SM-bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Effect of Physical Parameters on the Main Phase Transition of Supported Lipid Bilayers

Supported lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) were assembled by the vesicle fusion technique on mica and studied by temperature-controlled atomic force microscopy. The role of different physical parameters on the main phase transition was elucidated. Both mixed (POPE/POPG 3:1) and pure POPE bilayers were studied. By increasing the ionic strength of the solution and the incubation temperature, a shift from a decoupled phase transition of the two leaflets, to a coupled transition, with domains in register, was obtained. The observed behavior points to a modulation of the substrate/bilayer and interleaflet coupling induced by the environment and preparation conditions of supported lipid bilayers. The results are discussed in view of the role of different interactions in the system. The influence of the substrate on the lipid bilayers, in terms of interleaflet coupling, can also help us in understanding the possible effect that submembrane elements like the cytoskeleton might have on the structure and dynamics of biomembranes.

Hydrated cholesterol: Phospholipid domains probed by synchrotron radiation

X-ray scattering experiments on mixed films of cholesterol and phospholipids at air-water and Si solid-water interfaces were undertaken to glean information on pathological crystallization of cholesterol bilayers. Grazing-incidence X-ray diffraction patterns at the air-water interface of various cholesterol:dipalmitoyl-phosphatidylcholine (Ch:DPPC) monolayer mixtures compressed beyond monolayer collapse yielded the established 10 x 7.5 Ų Ch bilayer motif, for Ch:DPPC molar ratios higher than 2.5:1. Attempts to obtain a diffraction signal from various Ch:phospholipid film mixtures at the Si solid-water interface, indicative of the presence of the Ch bilayer motif, were unsuccessful. Only after removal of sufficient water from the cell was a weak diffraction signal obtained suggestive of a cholesterol film two bilayers thick. Off-specular X-ray reflectivity measurements made on a 1.75:1 mixture of Ch and bovine cardiac phosphatidylcholine (BCPC) deposited as a bilayer on a Si wafer and placed in a cell filled with water yielded positive results. The derived electron density profile showed the presence of a bilayer mixture consistent with a phase separation of cholesterol and BCPC, and possible formation of a crystalline cholesterol bilayer within the hydrated mixed bilayer, but not a proof thereof.