Construction of a DOPC/PSM/cholesterol phase diagram based on the fluorescence properties of trans-parinaric acid

Cell membranes have a nonhomogenous lateral organization. Most information about such nonhomogenous mixing has been obtained from model membrane studies where defined lipid mixtures have been characterized. Various experimental approaches have been used to determine binary and ternary phase diagrams for systems under equilibrium conditions. Such phase diagrams are the most useful tools for understanding the lateral organization in cellular membranes. Here we have used the fluorescence properties of trans-parinaric acid (tPA) for phase diagram determination. The fluorescence intensity, anisotropy, and fluorescence lifetimes of tPA were measured in bilayers composed of one to three lipid components. All of these parameters could be used to determine the presence of liquid-ordered and gel phases in the samples. However, the clearest information about the phase state of the lipid bilayers was obtained from the fluorescence lifetimes of tPA. This is due to the fact that an intermediate-length lifetime was found in samples that contain a liquid-ordered phase and a long lifetime was found in samples that contained a gel phase, whereas tPA in the liquid-disordered phase has a markedly shorter fluorescence lifetime. On the basis of the measured fluorescence parameters, a phase diagram for the 1,2-dioleoyl-sn-glycero-3-phosphocholine/N-palmitoyl sphingomyelin/cholesterol system at 23 °C was prepared with a 5 mol % resolution. We conclude that tPA is a good fluorophore for probing the phase behavior of complex lipid mixtures, especially because multilamellar vesicles can be used. The determined phase diagram shows a clear resemblance to the microscopically determined phase diagram for the same system. However, there are also significant differences that likely are due to tPA's sensitivity to the presence of submicroscopic liquid-ordered and gel phase domains.     

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.     

Interleaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations

In order to investigate experimentally inaccessible, molecular-level detail regarding interleaflet interaction in membranes, we have run an extensive series of coarse-grained molecular dynamics simulations of phase separated lipid bilayers. The simulations are motivated by differences in lipid and cholesterol composition in the inner and outer leaflets of biological membranes. Over the past several years, this phenomenon has inspired a series of experiments in model membrane systems which have explored the effects of lipid compositional asymmetry in the two leaflets. The simulations are directed at understanding one potential consequence of compositional asymmetry, that being regions of bilayers where liquid-ordered (L(o)) domains in one leaflet are opposite liquid-disordered (L(d)) domains in the other leaflet (phase asymmetry). The simulated bilayers are of two sorts: 1) Compositionally symmetric leaflets where each of the two leaflets contains an identical, phase separated (L(o)/L(d)) mixture of cholesterol, saturated and unsaturated phospholipid; and 2) Compositionally asymmetric leaflets, where one leaflet contains a phase separated (L(o)/L(d)) mixture while the other contains only unsaturated lipid, which on its own would be in the L(d) phase. In addition, we have run simulations where the lengths of the saturated lipid chains as well as the mole ratios of the three lipid components are varied. Collectively, we report on three types of interleaflet coupling within a bilayer. First, we show the effects of compositional asymmetry on acyl chain tilt and order, lipid rotational dynamics, and lateral diffusion in regions of leaflets that are opposite L(o) domains. Second, we show substantial effects of compositional asymmetry on local bilayer curvature, with the conclusion that phase separated leaflets resist curvature, while inducing large degrees of curvature in an opposing L(d) leaflet. Finally, in compositionally symmetric, phase separated bilayers, we find phase asymmetry (domain antiregistration) between the two leaflets occurs as a consequence of mismatched acyl chain-lengths in the saturated and unsaturated lipids.     

Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy

There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either l(o) or l(d) phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.     

Influence of cyclodextrins on the physical stability of DPPC-liposomes

This paper reports on the physical stability of DPPC-(dipalmitoyl phosphatidyl choline) liposomes in various aqueous dispersions and its control by uncharged polymers. The effect of natural (-, β-, γ-) cyclodextrins (CDs) on the stability of bare and polymer-bearing liposomes and also, the attachment of the CD molecules and the macromolecules, respectively, to the DPPC-bilayers of small unilamellar vesicles (SUV) were studied.

It was found that above a CD/DPPC ratio, each cyclodextrin caused a definite destruction in the phospholipid bilayers. The extent of membrane destabilization due to a cyclodextrin closely related to the amount of the CD molecules bound to the DPPC-bilayers.

The polymer-coated liposomes formulated by incorporating a dissolved homopolymer or copolymer into the phospholipid bilayer of the vesicles exhibited higher physical stability. Uncharged polymers effectively hindered the disintegration of the liposomal membranes brought about by the CD molecules. The polymer layers formed around the phospholipid bilayers ensured an enhanced steric stabilization for the DPPC-liposomes. Methylcellulose (MC) with high molecular mass and a polyvinyl alcohol-co-vinyl propional copolymer alike exhibited efficient stabilizing effect.     

Effects of isoflurane, halothane, and chloroform on the interactions and lateral organization of lipids in the liquid-ordered phase

The first quantitative insight has been obtained into the effects that volatile anesthetics have on the interactions and lateral organization of lipids in model membranes that mimic "lipid rafts". Specifically, nearest-neighbor recogntion measurements, in combination with Monte Carlo simulations, have been used to investigate the action of isoflurane, halothane, and chloroform on the compactness and lateral organization of cholesterol-rich bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the liquid-ordered (l(o)) phase. All three anesthetics induce a similar weakening of sterol-phospholipid association, corresponding to ca. 30 cal/mol of lipid at clinically relevant concentrations. Monte Carlo lattice simulations show that the lateral organization of the l(o) phase, under such conditions, remains virtually unchanged. In sharp contrast to their action on the l(o) phase, these anesthetics have been found to have a similar strengthening effect on sterol-phospholipid association in the liquid-disordered (l(d)) phase. The possibility of discrete complexes being formed between DPPC and these anesthetics and the biological relevance of these findings are discussed.     

Supported lipid bilayers lifted from the substrate by layer-by-layer polyion cushions on self-assembled monolayers

The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11-mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1-palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2–2.1)×10⁻⁹ cm²/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes.     

Nonintercalating nanosubstrates create asymmetry between bilayer leaflets

The physical properties of lipid bilayers can be remodeled by a variety of environmental factors. Here we investigate using molecular dynamics simulations the specific effects of nanoscopic substrates or external contact points on lipid membranes. We expose palmitoyl-oleoyl phosphatidylcholine bilayers unilaterally and separately to various model nanosized substrates differing in surface hydroxyl densities. We find that a surface hydroxyl density as low as 10% is sufficient to keep the bilayer juxtaposed to the substrate. The bilayer interacts with the substrate indirectly through multiple layers of water molecules; however, despite such buffered interaction, the bilayers exhibit certain properties different from unsupported bilayers. The substrates modify transverse lipid fluctuations, charge density profiles, and lipid diffusion rates, although differently in the two leaflets, which creates an asymmetry between bilayer leaflets. Other properties that include lipid cross-sectional areas, component volumes, and order parameters are minimally affected. The extent of asymmetry that we observe between bilayer leaflets is well beyond what has been reported for bilayers adsorbed on infinite solid supports. This is perhaps because the bilayers are much closer to our nanosized finite supports than to infinite solid supports, resulting in a stronger support-bilayer electrostatic coupling. The exposure of membranes to nanoscopic contact points, therefore, cannot be considered as a simple linear interpolation between unsupported membranes and membranes supported on infinite supports. In the biological context, this suggests that the exposure of membranes to nonintercalating proteins, such as those belonging to the cytoskeleton, should not always be considered as passive nonconsequential interactions.     

Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).     

Micropatterned composite membranes of polymerized and fluid lipid bilayers

Micropatterned composite membranes of polymerized and fluid lipid bilayers were constructed on solid substrates. Lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), and subsequent removal of nonreacted monomers by a detergent solution (0.1 M sodium dodecyl sulfate (SDS)) yielded a patterned polymeric bilayer matrix on the substrate. Fluid lipid bilayers of phosphatidylcholine from egg yolk (egg-PC) were incorporated into the lipid-free wells surrounded by the polymeric bilayers through the process of fusion and reorganization of suspended small unilamellar vesicles. Spatial distribution of the fluid bilayers in the patterned bilayer depended on the degree of photopolymerization that in turn could be modulated by varying the applied UV irradiation dose. The polymeric bilayer domains blocked lateral diffusion of the fluid lipid bilayers and confined them in the defined areas (corrals), if the polymerization was conducted with a sufficiently large UV dose. On the other hand, lipid molecules of the fluid bilayers penetrated into the polymeric bilayer domains, if the UV dose was relatively small. A direct correlation was observed between the applied UV dose and the lateral diffusion coefficient of fluorescent marker molecules in the fluid bilayers embedded within the polymeric bilayer domains. Artificial control of lateral diffusion by polymeric bilayers may lead to the creation of complex and versatile biomimetic model membrane arrays.     

Influence of perfluorinated compounds on the properties of model lipid membranes

The influence of selected perfluorinated compounds (PFCs), perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS), on the structure and organization of lipid membranes was investigated using model membranes-lipid monolayers and bilayers. The simplest model--a lipid monolayer--was studied at the air-water interface using the Langmuir-Blodgett technique with surface pressure and surface potential measurements. Lipid bilayers were characterized by NMR techniques and molecular dynamics simulations. Two phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), characterized by different surface properties have been chosen as components of the model membranes. For a DPPC monolayer, a phase transition from the liquid-expanded state to the liquid-condensed state can be observed upon compression at room temperature, while a DMPC monolayer under the same conditions remains in the liquid-expanded state. For each of the two lipids, the presence of both PFOA and PFOS leads to the formation of a more fluidic layer at the air-water interface. Pulsed field gradient NMR measurements of the lateral diffusion coefficient (DL) of DMPC and PFOA in oriented bilayers reveal that, upon addition of PFOA to DMPC bilayers, DL of DMPC decreases for small amounts of PFOA, while larger additions produce an increased DL. The DL values of PFOA were found to be slightly larger than those for DMPC, probably as a consequence of the water solubility of PFOA. Furthermore, 31P and 2H NMR showed that the gel-liquid crystalline phase transition temperature decreased by the addition of PFOA for concentrations of 5 mol % and above, indicating a destabilizing effect of PFOA on the membranes. Deuterium order parameters of deuterated DMPC were found to increase slightly upon increasing the PFOA concentration. The monolayer experiments reveal that PFOS also penetrates slowly into already preformed lipid layers, leading to a change of their properties with time. These experimental observations are in qualitative agreement with the computational results obtained from the molecular dynamics simulations showing a slow migration of PFCs from the surrounding water phase into DPPC and DMPC bilayers.     

Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration

An atomic force and fluorescence microscopy investigation of the structure of spin-coated lipid films is presented. In the surface of the dry film, lipids are found to orient in a conformation where acyl chains are pointing outward while laterally the individual layers of the multilamellar film exhibit a dewetting pattern similar to what is found in polymer thin films. Hydration of the film in liquid water promotes detachment of bilayers from the surface while a single membrane remains on the mica substrate. This supported membrane is highly uniform and defect-free as compared to supported membranes prepared by conventional methods. It is further demonstrated that supported membranes of binary lipid mixtures prepared by this method exhibit gel-fluid domain coexistence in accordance with expectations from the phase diagrams.     

Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers

The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83-101), a water-insoluble construct containing residues 89-101, and a water-soluble construct containing residues 89-101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10(4) M(-1) between the soluble peptide and phase-separated lipid bilayers and 10(3) M(-1) between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid-liquid melting temperature.     

The Effect of Cholesterol on Membrane Dynamics on Different Timescales in Lipid Bilayers from Fast Field‐Cycling NMR Relaxometry Studies of Unilamellar Vesicles

The general applicability of fast field‐cycling nuclear magnetic resonance relaxometry in the study of dynamics in lipid bilayers is demonstrated through analysis of binary unilamellar liposomes composed of 1,2‐dioleoyl‐sn‐glycero‐3‐posphocholine (DOPC) and cholesterol. We extend an evidence‐based method to simulating the NMR relaxation response, previously validated for single‐component membranes, to evaluate the effect of the sterol molecule on local ordering and dynamics over multiple timescales. The relaxometric results are found to be most consistent with the partitioning of the lipid molecules into affected and unaffected portions, rather than a single averaged phase. Our analysis suggests that up to 25 mol %, each cholesterol molecule orders three DOPC molecules, providing experimental backup to the findings of many molecular dynamics studies. A methodology is established for studying dynamics on multiple timescales in unilamellar membranes of more complex compositions.     

Interaction of propyl paraben with dipalmitoyl phosphatidylcholine bilayer: a differential scanning calorimetry and nuclear magnetic resonance study

The influence of the preservative, propyl paraben (PPB) on the biophysical properties of dipalmitoyl phosphatidyl choline (DPPC) vesicles, both in multilamellar vesicle (MLV) and unilamellar vesicle (ULV) forms, has been studied using DSC and (¹H and ³¹P) NMR. The mechanism by which PPB interacts with DPPC bilayers was found to be independent of the morphological organization of the lipid bilayer. Incorporation of PPB in DPPC vesicles causes a significant depression in the transition temperature and enthalpy of both the pre-transition (PT) and the gel to liquid crystalline transition. The presence of the PPB also reduces the co-operativity of these transitions. However, at high PPB concentration the PT disappears. DSC and NMR findings indicate that: (i) PPB is bound strongly to the lipid bilayer leading to increased headgroup fluidity due to reduced headgroup–headgroup interaction and (ii) the PPB molecules are intercalated between the DPPC polar headgroups with its alkyl chain penetrate into the co-operative region. MLV incorporated with high PPB concentration shows additional transitions whose intensity increases with increasing PPB concentration. This phase segregation observed could probably be due to co-existence of PPB-rich and PPB-poor phospholipid domains within the bilayers. The effect of inclusion of cholesterol in the PPB-free and PPB-doped DPPC dispersion was also studied. Equilibration studies suggest that PPB molecules are very strongly bound and remain intercalated between the polar headgroup for prolonged time.     

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.     

Membrane interactions of host-defense peptides studied in model systems

Host-defense, antibiotic peptides are believed to generate their cytolytic effects by interacting with the membranes of bacterial cells. Direct analyses of peptide interactions with real cellular membranes are difficult, however, due to the high complexity of physiological membranes. This review summarizes experimental work aiming to understand peptide-membrane interactions and their relationships with the peptides' biological actions using specific model systems. Varied model assemblies have been constructed that generally aim to mimic the fundamental lipid bilayer organization of the membrane. The model systems we will describe include multilamellar and unilamellar vesicles, planar lipid bilayers, lipid monolayers and micelles, and colorimetric biomimetic membranes. The different artificial models have facilitated examination of specific biological or chemical parameters affecting peptide action, for example the effect of membrane lipid composition on peptide affinities and membrane penetration, the relationship between membrane fluidity and peptide interactions, the conformations of active peptides, and other factors. We evaluate the strengths and limitations of the various approaches, and point to future directions in the field.     

Structural effects of a basic peptide on the organization of dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine membranes: a fluorescent resonance energy transfer study

We studied the effect of a model basic peptide, hexalysiltryptophan, on the organization of dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine unilamellar vesicles by means of fluorescent resonance energy transfer (FRET) between fluorescently labeled phospholipids. Several FRET theoretical models assuming different bilayer geometries and probe distributions were fitted to the time-resolved data. The experiments were carried out at two temperatures in different regions of the lipid mixture phase diagram. At 45 degrees C, the expected gel/fluid phase separation was verified by model fitting in peptide-free vesicles, which from the FRET approach means that domains are larger than approximately 200 A. No noticeable alteration of membrane organization was detected upon increasing the peptide concentration. At variance, for the single fluid phase at 60 degrees C, there was a large increase in FRET efficiency upon peptide addition to the lipid vesicles, mainly caused by peptide-induced vesicle aggregation. The system gradually changed from unilamellar lipid vesicles to a multibilayer geometry, and a limit lamellar repeat distance of approximately 57 A was recovered. Furthermore, no evidence for lateral domain formation on the FRET length scale was found at this temperature, the cationic peptide being only able to induce local lipid demixing, causing a short-range sequestration of 2-3 acidic lipids around each surface-adsorbed peptide.     

Molecular dynamics studies of the molecular structure and interactions of cholesterol superlattices and random domains in an unsaturated phosphatidylcholine bilayer membrane

The effect of the molecular organization of lipid components on the properties of the bilayer membrane has been a topic of increasing interest. Several experimental and theoretical studies have suggested that cholesterol is not randomly distributed in the fluid-state lipid bilayer but forms nanoscale domains. Several cholesterol-enriched nanodomain structures have been proposed, including rafts, regular or maze arrays, complexes, and superlattices. At present, the molecular mechanisms by which lipid composition influences the formation and stability of lipid nanodomains remain unclear. In this study, we have used molecular dynamics (MD) simulations to investigate the effects of the molecular organization of cholesterol--superlattice versus random--on the structure of and interactions between lipids and water in lipid bilayers of cholesterol and 1-palmitoyl-2-oleoylphosphatidylcholine (cholesterol/POPC) at a fixed cholesterol mole fraction of 0.40. On the basis of four independent replicates of 200-ns MD simulations for a superlattice or random bilayer, statistically significant differences were observed in the lipid structural parameters, area per lipid, density profile, and acyl chain order profile, as well as the hydrogen bonding between various pairs (POPC and water, cholesterol and water, and POPC and cholesterol). The time evolution of the radial distribution of the cholesterol hydroxy oxygen suggests that the lateral distribution of cholesterol in the superlattice bilayer is more stable than that in the random bilayer. Furthermore, the results indicate that a relatively long simulation time, more than 100 ns, is required for these two-component bilayers to reach equilibrium and that this time is influenced by the initial lateral distribution of lipid components.     

Filipin orientation revealed by linear dichroism. Implication for a model of action

The organization of the polyene antibiotic filipin in membranes containing cholesterol is a controversial matter of debate. Two contradictory models exist, one suggesting a parallel and the other perpendicular organization of filipin with respect to the plane of the membrane. UV-vis linear dichroism, ATR-FTIR, and fluorescence anisotropy decay techniques were combined to study the orientation of filipin in model systems of membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) with and without cholesterol. Filipin's orientation is determined by the presence/absence of cholesterol when it is inserted in gel crystalline phase model membranes. When cholesterol (33%) is present in DPPC bilayers, filipin stands perpendicular to the membrane surface as expected in "pore-forming" models. At variance, absence of cholesterol leaves filipin in an essentially random organization in the lipidic matrix. In liquid crystalline phase bilayers (POPC) filipin's orientation is perpendicular to the membrane surface even in absence of cholesterol. Thus filipin's activity/organization depends not only on cholesterol presence but also in the lipid phase domain it is inserted in. These findings were combined with spectroscopy and microscopy data in the literature, solving controversial matters of debate.