Different oxidized phospholipid molecules unequally affect bilayer packing

The aim of this study was to gain more detailed knowledge about the effect of the presence of defined oxidized phospholipid molecules in phospholipid bilayers. After chromatographic and mass spectrometry analysis, the previously used product of the Fenton reaction with unsaturated lecithins proved to consist of a plethora of oxidatively modified lecithins, unuseful either for the detailed study of the effects brought about in the bilayer or as the source of defined oxidized phospholipid molecules. The latter, particularly 2-(ω-carboxyacyl)- and 2-(n-hydroperoxyacyl)-lecithins, can be more conveniently prepared by chemical or enzymatic synthesis rather than by chemical or physical oxidation. The effect of those molecules and of commercially available 12-hydroxy-stearic and dodecanedioic acid was studied in planar supported phospholipid bilayers (SPBs) by use of EPR spectrometry. The SPBs also contained 2-(5-doxylstearoyl)-lecithin as the spin probe, and the EPR spectral anisotropy loss, indicative of bilayer disordering, was measured as a function of the molar percentage of oxidized lipid. Most oxidized lipid molecules examined in this study were able to induce bilayer disordering, while hydroperoxyl group-bearing acyl chains appeared to be much less effective. It is concluded that the effects of different oxidized phospholipids on phospholipid bilayer structure cannot be generalized, as happens with batch-oxidized phospholipids, and that the use of defined oxidized phospholipid molecular species for membrane oxidative stress guarantees a more reliable and detailed response.     

EPR studies of phospholipid bilayers after lipoperoxidation. 1. Inner molecular order and fluidity gradient

The molecular order of fatty acyl chains in oriented lipid bilayers on solid support (SPB), made of either natural or synthetic phospholipids oxidized by Fenton reagent and probed with spin labeled lecithin (5-DSPC) was studied by means of EPR spectrometry. Phospholipids (ASPC, EYPC, mitochondrial extract) were oxidized as either aqueous buffer/methanol dispersions or reverse-phase evaporation vesicles (REV) suspensions. Oxidation was preliminarily revealed both by assaying MDA and by detecting conjugated dienes. Oxidized phospholipid species was quantified by preparative TLC. The degree of order in oriented lipid bilayers of samples containing oxidized phospholipids was estimated by the loss of EPR spectral anisotropy, and an empirical index of the related bilayer disorder was calculated from the second derivative spectra. Bilayers made of each non-oxidized phospholipid species from either ethanol solutions or REV suspensions showed the highest anisotropy, while the increasing presence of oxidized lipids in the samples resulted in progressive loss of EPR spectral anisotropy. In contrast, vesicles containing 40% of the oxidized species maintained an unaltered fluidity gradient, while REV formation was hindered by oxidized phospholipid percentages higher than 45% for ASPC and EYPC, and 35% for Mitochondrial lipids (MtL). It is concluded that the early stages of lipoperoxidation bring about disordering of the phospholipid bilayer interior rather than fluidity alterations, and that prolonged oxidation may result in loss of structural and chemical properties of the bilayer until the structure no longer holds.     

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, β-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.     

Comparative 5-doxylstearoyllecithin and 3-doxylcholestane EPR spin labeling study of phospholipid bilayer perturbation by different oxidized lecithin species

A 3-doxylcholestane spin label was employed in addition to 5-doxylstearoyl lecithin for a more detailed study of the different effects exerted by variously oxidized lecithins on fatty acid alignment in phospholipid planar bilayers. Either spin label was enclosed in oriented PLPC planar samples also containing in turn a variety of conjugated-dienes lecithins and cleaved chain lecithins, in order to monitor EPR spectral angular dependence loss. Data obtained by use of arachidonoyl-hydroxystearoyl-PC and palmitoyl-hydroxylinoleoyl-PC confirm that the 5-DSPC nitroxide ring almost completely retains its orientation in CD-PCs-containing planar membranes, in contrast with angular dependence loss observed in the presence of the CC-PC molecular species palmitoyl-oxononanoyl-PC and palmitoyl-oxovaleroyl-PC, already seen with cleaved-chain palmitoyl-glutaroyl-PC and palmitoyl-azelaoyl-PC. However, the 3-DC nitroxide ring also loses its orientation with CD-PCs, in addition to being disoriented by cleaved chain-lecithins, similarly to 5DSPC. Joint information from the two spin labels will help to clarify whether OXPC-related disordering involved the whole bilayer structure or only the hydrophobic core. In addition, the propensity of different OXPCs to form bilayer vesicles in water suspension was also determined by Sepharose 4B gel-chromatography. The results suggest that CD-PCs might yield SPB bilayer structures with a disordered hydrophobic core, while pure CC-PC more probably forms non-bilayer disordered structures, possibly micelles or mixed micelle/bilayers.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.     

Spin labeling EPR studies of the properties of oxidized phospholipid-containing lipid vesicles

This study aims at characterizing the structure and some properties of phospholipid multi-lamellar vesicles (MLVs) containing the oxidized species γ-palmitoyl-β-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin (HPPLPC), γ-palmitoyl-β-(9-hydroxy-10,12-octadecanedienoyl)-lecithin (HOPLPC), γ-palmitoyl-β-glutaroyl-lecithin (GlPPC) and γ-palmitoyl-β-azelaoyl-lecithin (AzPPC). Sepharose 4B gel-chromatography was used to ensure and check that only MLVs are used in EPR measurements. Gel-solid to gel-liquid transition temperature (T_m), lateral phase separation, fluidity gradient and polarity profile were studied by use of EPR spectroscopy of enclosed n-doxylstearoyl lecithin spin labels. Contrarily to conjugate dienes and normal phospholipids, pure carboxyacyl species yielded aqueous suspensions showing gel-chromatography elution profile resembling that of lysolecithin micelles. Conjugate dienes/DPPC MLVs showed lateral phase separation at room temperature and T_m value lower than pure DPPC MLVs. Pure conjugate dienes MLVs resembled more PLPC MLVs and displayed free miscibility with PLPC in mixed MLVs. Pure HPPLPC MLV bilayer appeared to be slightly more rigid, while that of HOPLPC and the polarity profile of MLVs made of the pure conjugate dienes species were similar to those of normal PLPC. It is concluded that carboxyacyl lecithins in MLVs tend to disrupt vesicle structure, while conjugated dienes lecithins are more able to affect some physical properties of the bilayer, and that DPPC in MLVs enhances these effects while PLPC shows a better compatibility with the lipoperoxides.     

Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations

Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophore’s immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.     

Photolabelling of D-beta-hydroxybutyrate apodehydrogenase with azidoaryl phospholipids

Two synthetic photoactive azidoarylphosphatidylcholines were used to investigate the level of interaction between D-beta-hydroxybutyrate apodehydrogenase (apoBDH), an amphipathic membrane protein, with the hydrophobic domain of phospholipids. The two synthetic lecithins, PL I (1-myristoyl-2-12-N-(4-azido-2-nitrophenyl) aminododecanoyl-sn-glycero-3-phosphocholine) and PL II (1-myristoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-phosphocholine), are able to reactivate the non-active purified apoBDH as well as the non-photoactive homologs, indicating that the photoreactive chemical groups are without effect on the cofactor properties of phosphatidylcholine. Photoirradiation of reconstituted complexes between phospholipid containing azidoaryllecithin and apoBDH leads to a covalent binding of some synthetic lecithin molecules on the protein. The labelling, about 3 times higher with PL II than with PL I, suggests that the area of interacting domain of BDH with the hydrophobic moiety of phospholipid is more important at or near the surface of the lipid bilayer than in the inner part. This approach is further demonstration that BDH is an integral protein.     

Muscovite (mica) allows the characterisation of supported bilayers by ellipsometry and confocal fluorescence correlation spectroscopy

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.     

Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.     

Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

Effect of doxorubicin on the order of the acyl chains of anionic and zwitterionic phospholipids in liquid-crystalline mixed model membranes: absence of drug-induced segregation of lipids into extended domains.

We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.     

Lipid composition modulates the interaction of peptides deriving from herpes simplex virus type I glycoproteins B and H with biomembranes

Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626-644 and gB632-650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626-644 localizes close to the hydrophilic bilayer surface, while gB632-650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632-650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626-644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632-650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer.     

Interaction between Alzheimer's Abeta(25-35) peptide and phospholipid bilayers: the role of cholesterol

There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of beta-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Abeta(1-42), i.e., Abeta(25-35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Abeta(25-35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Abeta(25-35).     

Small angle x-ray scattering studies of magnetically oriented lipid bilayers.

Magnetically oriented lipid/detergent bilayers are potentially useful for studies of membrane-associated molecules and complexes using x-ray scattering and nuclear magnetic resonance (NMR). To establish whether the system is a reasonable model of a phospholipid bilayer, we have studied the system using x-ray solution scattering to determine the bilayer thickness, interparticle spacing, and orientational parameters for magnetically oriented lipid bilayers. The magnetically orientable samples contain the phospholipid L-alpha-dilauroylphosphatidylcholine (DLPC) and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) in a 3:1 molar ratio in 70% water (w/v) and are similar to magnetically orientable samples used as NMR media for structural studies of membrane-associated molecules. A bilayer thickness of 30 A was determined for the DLPC/CHAPSO particles, which is the same as the bilayer thickness of pure DLPC vesicles, suggesting that the CHAPSO is not greatly perturbing the lipid bilayer. These data, as well as NMR data on molecules incorporated in the oriented lipid particles, are consistent with the sample consisting of reasonably homogeneous and well dispersed lipid particles. Finally, the orientational energy of the sample suggests that the size of the cooperatively orienting unit in the samples is 2 x 10(7) phospholipid molecules.     

Molecular dynamics simulation studies of lipid bilayer systems

The main structural element of biological membranes is a liquid-crystalline lipid bilayer. Other constituents, i.e. proteins, sterols and peptides, either intercalate into or loosely attach to the bilayer. We applied a molecular dynamics simulation method to study membrane systems at various levels of compositional complexity. The studies were started from simple lipid bilayers containing a single type phosphatidylcholine (PC) and water molecules (PC bilayers). As a next step, cholesterol (Chol) molecules were introduced to the PC bilayers (PC-Chol bilayers). These studies provided detailed information about the structure and dynamics of the membrane/water interface and the hydrocarbon chain region in bilayers built of various types of PCs and Chol. This enabled studies of membrane systems of higher complexity. They included the investigation of an integral membrane protein in its natural environment of a PC bilayer, and the antibacterial activity of magainin-2. The latter study required the construction of a model bacterial membrane which consisted of two types of phospholipids and counter ions. Whenever published experimental data were available, the results of the simulations were compared with them.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.