Structure and thermotropic phase behavior of fluorinated phospholipid bilayers: a combined attenuated total reflection FTIR spectroscopy and imaging ellipsometry study

Lipid bilayers consisting of lipids with terminally perfluoroalkylated chains have remarkable properties. They exhibit increased stability and phase-separated nanoscale patterns in mixtures with nonfluorinated lipids. In order to understand the bilayer properties that are responsible for this behavior, we have analyzed the structure of solid-supported bilayers composed of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and of a DPPC analogue with 6 terminal perfluorinated methylene units (F6-DPPC). Polarized attenuated total reflection Fourier-transform infrared spectroscopy indicates that for F6-DPPC, the tilt of the lipid acyl chains to the bilayer normal is increased to 39 degrees as compared to 21 degrees for native DPPC, for both lipids in the gel phase. This substantial increase of the tilt angle is responsible for a decrease of the bilayer thickness from 5.4 nm for DPPC to 4.5 nm for F6-DPPC, as revealed by temperature-controlled imaging ellipsometry on microstructured lipid bilayers and solution atomic force microscopy. During the main phase transition from the gel to the fluid phase, both the relative bilayer thickness change and the relative area change are substantially smaller for F6-DPPC than for DPPC. In light of these structural and thermotropic data, we propose a model in which the higher acyl-chain tilt angle in F6-DPPC is the result of a conformational rearrangement to minimize unfavorable fluorocarbon-hydrocarbon interactions in the center of the bilayer due to chain staggering.     

Cholesterol induced suppression of large swelling of water layer in phosphocholine floating bilayers

The effect of the addition of 10 mol % cholesterol to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) floating bilayers has been investigated by means of neutron reflectivity measurements. The large swelling of the water layer between the two bilayers found in pure phosphocholine systems around the lipid main phase transition is greatly reduced in the 9:1 DPPC:cholesterol mixture. The analysis of the structure of the bilayer reveals that in the gel phase cholesterol induces the presence of a high rms roughness that disappears in the fluid phase.     

Role of nanomechanical properties in the tribological performance of phospholipid biomimetic surfaces

The role of phospholipid bilayers in controlling and reducing frictional forces between biological surfaces is investigated by three complementary experiments: friction forces are measured using a homemade tribometer, mechanical resistance to indentation is measured by AFM, and lipid bilayer degradation is controlled in situ during friction testing using fluorescence microscopy. DPPC lipid bilayers in the solid phase generate friction coefficients as low as 0.002 (comparable to that found for cartilage) that are stable through time. DOPC bilayers formed by the vesicle fusion method or the adsorption of mixed micelles generate higher friction coefficients. These coefficients increased through time, during which the bilayers degraded. The friction coefficient is correlated with the force needed to penetrate the bilayer with the AFM tip. With only one bilayer in the contact region, the friction increased to a similar value of about 0.08 for the DPPC and DOPC. Our study therefore shows that good mechanical stability of the bilayers is essential and suggests that the low friction coefficient is ensured by the hydration layers between adjacent lipid bilayers.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Effect of low amounts of cholesterol on the swelling behavior of floating bilayers

The effect of the addition of 1, 2, 4, and 6 mol % cholesterol to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) floating bilayers has been investigated by neutron reflectivity. All samples exhibited fully stable and reversible gel and fluid phases. Around the main lipid phase transition temperature, DPPC double bilayers exhibit large increases in the water layer separating the bilayers and the upper bilayer roughness. The inclusion of low amounts of cholesterol reduced the swelling of the water layer between the bilayers and the upper bilayer roughness and progressively widened the temperature range over which swelling occurs. Results from asymmetric bilayers are also reported. A higher amount of cholesterol in the lower bilayer induces a smaller swelling of the water layer between the bilayers than in the symmetric case. Finally, the effect of the inclusion of a leaflet of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was investigated. The presence of a leaflet with a higher gel-transition temperature (T(m)) modifies the phase behavior of the lower T(m) leaflet.     

The interaction of a peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with DPPC model membranes: a DSC study

Depending on their hydrophobicity, peptides can interact differently with lipid membranes inducing dramatic modifications into their host systems. In the present paper, the interaction of a synthetic peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes has been investigated by differential scanning calorimetry (DSC), adopting three different experimental approaches. In the first, the peptide is forced to be included into the hydrocarbon region of the lipid bilayer, by codissolving it with the lipid giving rise to mixed multilamellar vesicles–peptide systems; in the second, this system is passed through an extruder, thus producing large unilamellar vesicles–peptide systems; in the third, it is allowed to interact with the external surface of the membrane.

The whole of the DSC results obtained have shown that the incorporation of the peptide into the lipid bilayer by means of the first method induces a decrease in the enthalpy of the gel–liquid crystal transition of the membrane and a shift of the transition to the lower temperatures, thus resembling, in spite of its prevalently hydrophilic nature, the behavior of transbilayer hydrophobic peptides. The extrusion of these systems creates unilamellar vesicles free of peptides but of smaller size as evidenced by the decreased cooperativity of the transition. The peptide, added externally to the DPPC model membrane, has no effect on the phase behavior of the bilayer.

These findings suggest that the effect of the interaction of scrambled hydrophobic/hydrophilic peptides into lipid bilayers strongly affects the thermotropic behavior of the host membrane depending on the preparation method of the lipid/peptide systems. The whole of the results obtained in the present paper can be useful in approaching studies of bioactive peptides/lipids systems.     

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.     

UV-induced bursting of cell-sized multicomponent lipid vesicles in a photosensitive surfactant solution

We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (L(o)) phase and a DOPC-rich liquid-disordered (L(d)) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Y(burst)), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Y(burst) decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Y(burst) increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of L(o) phase (with increasing [Chol] ≥ 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Y(burst) = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.     

Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles

We have investigated the adsorption of phospholipid mixtures using neutron reflection. Small sonicated unilamellar vesicles (SUV) composed of DOPC and d(62)-DPPC were incubated at 50 degrees C in contact with a silica surface using a method commonly employed to form supported model membranes. The composition of the mixed supported bilayer was found to be substantially different from that of the bulk vesicles in a direction indicating a higher affinity of DPPC for the silica surface. Formation of an asymmetric bilayer arrangement was also discovered in all the cases studied. DPPC tended to dominate the composition of the leaflet next to silica, while the outer leaflet was generally closer to the bulk composition. The supported bilayers also exhibited increasing interfacial roughness in the outer membrane leaflet in the region of the DOPC-DPPC gel-liquid immiscibility region. To our knowledge, this is the first time that both the structure and the absolute composition of a mixed-lipid supported bilayer have been resolved, and the results raise a number of questions regarding the adsorption of vesicles and the properties of supported bilayers, which are discussed in terms of the bulk phase diagram of DOPC and DPPC.     

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.     

Methotrexate interaction with a lipid membrane model of DPPC

Dipalmitoylphosphatidylcholine (DPPC) liposomes were employed as membrane models for the investigation of the interaction occurring between methotrexate (MTX) and bilayer lipid matrix. Liposomes were obtained by hydrating a lipid film with 50 mM Tris buffer (pH 7.4). The differential scanning calorimetry (DSC) evaluation of the thermotropic parameters associated with the phase transitions of DPPC liposomes gave useful information about the kind of drug-membrane interaction. The results showed an electrostatic interaction taking place with the negatively charged molecules of MTX and the phosphorylcholine head groups, constituting the outer part of DPPC bilayers. No interaction with the hydrophobic phospholipid bilayer domains was detected, revealing a poor capability of MTX to cross through lipid membranes to reach the interior compartment of a lipid bounded structure. These findings correlate well within vitro biological experiments on MTX cell susceptibility.     

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.     

Phospholipid bilayers as biomembrane-like barriers in layer-by-layer polyelectrolyte films

Dipalmitoylphosphatidylcholine (DPPC) bilayer was created on the surface of an exponentially growing poly(glutamic acid)/poly(lysine) (PGA/PLL) layer-by-layer polyelectrolyte film. The lipid bilayer decreased the surface roughness of the polyelectrolyte film. The layer-by-layer construction of the polyelectrolyte film could be continued on the top of the DPPC layer. The lipid bilayer, however, formed a barrier in the interior of the polyelectrolyte film, which blocked the diffusion (a prerequisite for exponential growth) of the polyelectrolytes. Thus, a new growth regime started in the upper part of the polyelectrolyte film, which was added to embed the DPPC bilayer. The structure and the dynamics of the DPPC bilayer on the polyelectrolyte film surface remained similar to that of its hydrated multibilayers, except that the phase transition became wider. In the case of embedded DPPC bilayers, in addition, the phase-transition temperature also decreased. This is the result of interactions with the nonconcerted movements of the barrier-separated lower and higher parts of the polyelectrolyte film. Gramicidin A (GRA) as a model of lipid-soluble peptides and proteins was successfully incorporated into such DPPC films. The DPPC films, either with or without GRA, were remarkably stable; as many heating-cooling cycles to measure phase transition could be carried out without visible alterations as wanted.     

Composition of supported model membranes determined by neutron reflection

We have investigated the formation of supported bilayers by coadsorption of dipalmitoyl phosphatidylcholine (DPPC) with the nonionic surfactant beta-D-dodecyl maltoside. The adsorption of mixed phospholipid-surfactant micelles on hydrophilic silica surfaces at 25 degrees C was followed as a function of bulk concentration by neutron reflection. Using chain-deuterated d(25)-beta-D-dodecyl maltoside and d(62)-DPPC, we demonstrate that it is possible to determine the composition of the bilayers at each stage of a sequential dilution process, which enriches the adsorbed layer in phospholipid and leads to complete elimination of the surfactant. The final supported bilayers have thicknesses of 51 +/- 3 A and are stable to heating to 37 degrees C once all surfactant has been removed, and the structures agree well with other published data on DPPC supported bilayers. The coadsorption of cholesterol in a DPPC-surfactant mixture was also achieved, and the location and volume fraction of cholesterol in the DPPC bilayer was determined. Cholesterol is located in a 18 +/- 1 A thick layer below the lipid headgroup region and leads to an increased bilayer thickness of 58 +/- 2 A at 26 mol % of cholesterol.     

Heterogeneous molecular distribution in supported multicomponent lipid bilayers

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.     

Thermotropic phase transition in soluble nanoscale lipid bilayers

The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are water-soluble, monodisperse, self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 A and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 A, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs, and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide the basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.     

Direct submolecular scale imaging of mesoscale molecular order in supported dipalmitoylphosphatidylcholine bilayers

Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microscopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.     

Vesicle and bilayer formation of diphytanoylphosphatidylcholine (DPhPC) and diphytanoylphosphatidylethanolamine (DPhPE) mixtures and their bilayers' electrical stability

Lipid bilayers are of interest in applications where a cell membrane mimicking environment is desired. The performance of the lipid bilayer is largely dependent on the physical and chemical properties of the component lipids. Lipid bilayers consisting of phytanoyl lipids have proven to be appropriate choices since they exhibit high mechanical and chemical stability. In addition, such bilayers have high electrical resistances. Two different phytanoyl lipids, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and various combinations of the two have been investigated with respect to their behavior in aqueous solutions, their interactions with solid surfaces, and their electrical stability. Dynamic light scattering, nuclear magnetic resonance diffusion, and cryogenic transmission electron microscopy measurements showed that pure DPhPC as well as mixtures of DPhPC and DPhPE consisting of greater than 50% (mol%) DPhPC formed unilamellar vesicles. If the total lipid concentration was greater than 0.15g/l, then the vesicles formed solid-supported bilayers on plasma-treated gold and silica surfaces by the process of spontaneous vesicle adsorption and rupture, as determined by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. The solid-supported bilayers exhibited a high degree of viscoelasticity, probably an effect of relatively high amounts of imbibed water or incomplete vesicle fusion. Lipid compositions consisting of greater than 50% DPhPE formed small flower-like vesicular structures along with discrete liquid crystalline structures, as evidenced by cryogenic transmission electron microscopy. Furthermore, electrophysiology measurements were performed on bilayers using the tip-dip methodology and the bilayers' capacity to retain its electrical resistance towards an applied potential across the bilayer was evaluated as a function of lipid composition. It was shown that the lipid ratio significantly affected the bilayer's electrical stability, with pure DPhPE having the highest stability followed by 3DPhPC:7DPhPE and 7DPhPC:3DPhPE in decreasing order. The bilayer consisting of 5DPhPC:5DPhPE had the lowest stability towards the applied electrical potential.     

Membrane resistance to Triton X-100 explored by real-time atomic force microscopy

Lateral segregation of lipids and proteins in biological membranes leads to the formation of detergent-resistant domains, also called "rafts". Understanding the mechanisms governing the biomembrane's resistance to solubilization by detergents is crucial in biochemical research. Here, we used real-time atomic force microscopy (AFM) imaging to visualize the behavior of a model supported lipid bilayer in the presence of different Triton X-100 (TX-100) concentrations. Mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) supported bilayers were prepared by vesicle fusion. Real-time AFM imaging revealed that, at concentrations below the critical micelle concentration (CMC), TX-100 did not solubilize the bilayer, but the DPPC domains were eroded in a time-dependent manner. This effect was attributed to the DPPC molecular packing disorganization by the detergent starting from the DOPC/DPPC interface. Just above the CMC, the detergent led to a complete solubilization of the DOPC matrix, leaving the DPPC domains unaltered. At higher TX-100 concentrations, the DOPC was also immediately removed just after detergent addition, and the DPPC domains remaining on the mica surface appeared to be more swollen and were gradually solubilized. This progressive solubilization of the DPPC remaining phase did not start at the edge of the domains but from holes appearing and expanding at the center of DPPC patches. The swelling of the DPPC domains was directly correlated with TX-100 concentration above the CMC and with detergent intercalation between DPPC molecules. We are convinced that this approach will provide a key system to elucidate the physical mechanisms of membrane solubilization by nonionic detergents.     

Self-consistent mean-field model based on molecular dynamics: application to lipid-cholesterol bilayers

We have developed a dynamic self-consistent mean-field model, based on molecular-dynamics simulations, to study lipid-cholesterol bilayers. In this model the lipid bilayer is represented as a two-dimensional lattice field in the lipid chain order parameters, while cholesterol molecules are represented by hard rods. The motion of rods in the system is continuous and is not confined to lattice cells. The statistical mechanics of chain ordering is described by a mean field derived from an extension of a model due to Marcelja. The time evolution of the system is governed by stochastic equations. The ensemble of chain configurations required in partition sums, and the energies of interaction, are taken from atomistic level molecular-dynamics simulations of lipid bilayers. The model allows us to simulate systems 500 nm in lateral size for 20 micros time scales, or greater. We have applied the model to dipalmitoyl-phosphatidylcholine-cholesterol (Chol) bilayers at 50 degrees C for Chol concentrations between 2% and 33%. At low concentrations of Chol (2%-4%), the model predicts the formation of isolated clusters of Chol surrounded by relatively ordered lipid chains, randomly dispersed in the disordered bilayer. With increasing Chol composition, regions of Chol-induced order begin to overlap. Starting from about 11% Chol this ordering effect becomes system wide and regions unaffected by Chol are no longer detectable. From the analysis of properties of the model we conclude that the change in lipid chain order with increasing Chol concentration is continuous over the 20-mus scale of the simulations. We also conclude that at 50 degrees C no large-scale Chol-rich and Chol-depleted coexisting phase-separated regions form at any concentration. At no point in any of the simulations do we observe a higher degree of lateral organization, such as Chol-based superlattice structures.