Structures of amphotericin B-cholesterol complex.

The structures of amphotericin B-cholesterol complex that forms a channel in a lipid membrane were analysed by molecular mechanics calculations. The symmetric complex consisting of eight rigid antibiotic and cholesterol molecules was considered. The presence of a continuous set of low-energy states of the complex with different values of the channel diameter was shown. These states are characterized by significant tilt of the amphotericin planes to the radial axis of the channel and by strong interaction between the charged ammonium and carboxyl groups of the antibiotic. Changes of the channel diameter may result in changes in pore permeability.     

Bacteriorhodospin: a trans-membrane pump containing alpha-helix.

The probable arrangement of the bacteriorhodopsin molecules in the purple membrane of Halobacterium halobium is in clusters of three, with a 3-fold axis at the centre of each cluster; the axis is at right angles to the plane of the membrane. The proposed arrangement and the results of model calculations together indicate that each protein molecule spans the entire thickness of the membrane. An earlier proposal for the structure had the protein molecules in two layers, and it was symmetric in projection onto the profile-axis. This model is now rejected since it would be difficult to account for the recently discovered function of pumping protons. There remains a discrepancy in that the calculated number of protein molecules in the unit-cell is 3.4 compared to the three expected.The X-ray diffraction patterns from dispersions of the lipids extracted from the red and purple membranes of H. halobium are described.Model calculations are reported, which are based on the bilayer profile calculated for the extracted lipids and on two simple profiles for the protein. The calculations favour a structure for the purple membrane having the lipid molecules in two layers, as in a bilayer, although there may be more of the lipid on one side of the membrane than on the other. Assuming bilayer structure, the diffraction nearest the centre of the oriented pattern suggests that the lipid molecules may be located mainly in a few discrete regions, roughly 20 Å across, between the protein molecules. An uninterrupted monolayer of the lipid on one surface of a sheet of the protein molecules gives poor agreement with the observed profile-diffraction.The X-ray diffraction pattern from the oriented membranes suggested α-helix in the bacteriorhodopsin, and this has been confirmed by recording a 1.5 Å-reflection oriented on the profile-axis. There appear to be at least two segments of α-helix, which are somewhat inclined to one another, and the two may be packed together. Prominent diffraction on the in-plane axis near 10 Å is consistent with the segments lying more or less perpendicular to the plane of the membrane.     

Quantitative characterization of the lateral distribution of membrane proteins within the lipid bilayer.

The dependence of the lateral distribution of membrane proteins on the size, protein/lipoid molar ratio, and the magnitude of the interaction potentials has been investigated by computer modeling protein-lipid distributions with Monte Carlo calculations. These results have allowed us to develop a quantitative characterization of the distribution of membrane proteins and to correlate these distributions with experimental observables. The topological arrangement of protein domains, protein plus annular lipid domains, and free lipid domains is described in terms of radial distribution, pair connectedness, and cluster distribution functions. The radial distribution functions are used to measure the distribution of intermolecular distances between protein molecules, whereas the pair connectedness functions are used to estimate the physical extension of compositional domains. It is shown that, at characteristic protein/lipid molar ratios, previously isolated domains become connected, forming domain networks that extend over the entire membrane surface. These changes in the lateral connectivity of compositional domains are paralleled by changes in the calculated lateral diffusion coefficients and might have important implications for the regulation of diffusion controlled processes within the membrane.     

Lipid-protein interactions

Intrinsic membrane proteins are solvated by a shell of lipid molecules interacting with the membrane-penetrating surface of the protein; these lipid molecules are referred to as annular lipids. Lipid molecules are also found bound between transmembrane α-helices; these are referred to as non-annular lipids. Annular lipid binding constants depend on fatty acyl chain length, but the dependence is less than expected from models based on distortion of the lipid bilayer alone. This suggests that hydrophobic matching between a membrane protein and the surrounding lipid bilayer involves some distortion of the transmembrane α-helical bundle found in most membrane proteins, explaining the importance of bilayer thickness for membrane protein function. Annular lipid binding constants also depend on the structure of the polar headgroup region of the lipid, and hotspots for binding anionic lipids have been detected on some membrane proteins; binding of anionic lipid molecules to these hotspots can be functionally important. Binding of anionic lipids to non-annular sites on membrane proteins such as the potassium channel KcsA can also be important for function. It is argued that the packing preferences of the membrane-spanning α-helices in a membrane protein result in a structure that matches nicely with that of the surrounding lipid bilayer, so that lipid and protein can meet without either having to change very much.     

Oxygen profiles in membranes

Transmembrane profiles of molecular oxygen in lipid bilayers are not only significant for membrane physiology and pathology, but also are essential to the determination of membrane protein structure by site-directed spin labeling. Oxygen profiles obtained with spin-labeled lipid chains have a Boltzmann sigmoidal dependence on the depth into each lipid leaflet, which represents a two-compartment distribution between outer and inner regions of the membrane, with a transfer free energy that depends linearly on distance from the dividing planes. Transmembrane profiles for intramembrane polarity, and for water penetration into the membrane, have an identical form, but are of the reverse sign. Comparison with recently published oxygen profiles from a site-specifically spin-labeled alpha-helical transmembrane peptide validates the use of spin-labeled lipids for all these profiles and provides the necessary bridge to generate the full bilayer from a single lipid leaflet.     

Molecular packing parameters of bipolar lipids.

The bipolar lipid fractions extracted from the thermophilic archaeobacterium Sulfolobus solfataricus have different chemical structures and geometrical shapes. The conditions which lead to the formation of vesicles were investigated in order to study the self-assembly of these molecules. Such conditions are fulfilled when an appropriate mixture of two different molecular species (both bipolar or bipolar and monopolar) is used. According to the theory introduced by Israelachvili and co-workers, lipid self-assembly results from the balance of interaction free energy, entropy and molecular geometry. We have shown that this theory can be extended to bipolar lipids, in spite of their more complex nature, and the experimental results obtained combining 1H-NMR, light scattering and entrapped volume techniques closely match theoretical expectations. To carry out calculations, it was necessary to introduce hypotheses about the disposition of bipolar molecules in the vesicle membrane. These hypotheses have been tested indirectly by measuring the transport properties mediated by carriers or channels, whose transport mechanism can be considered to be a probe of the membrane structure.     

How lipids and proteins interact in a membrane: a molecular approach

Membrane proteins in a biological membrane are surrounded by a shell or annulus of 'solvent' lipid molecules. These lipid molecules in general interact rather non-specifically with the protein molecules, although a few 'hot-spots' may be present on the protein where anionic lipids bind with high affinity. Because of the low structural specificity of most of the annular sites, the composition of the lipid annulus will be rather similar to the bulk lipid composition of the membrane. The structures of the solvent lipid molecules are important in determining the conformational state of a membrane protein, and hence its activity, through charge and hydrogen bonding interactions between the lipid headgroups and residues in the protein, and through hydrophobic matching between the protein and the surrounding lipid bilayer. Evidence is also accumulating for the presence of 'co-factor' lipid molecules binding with high specificity to membrane proteins, often between transmembrane alpha-helices, and often being essential for activity.     

Molecular mechanisms of calcium-induced membrane fusion

We have reviewed studies on calcium-induced fusion of lipid bilayer membranes and the role of synexin and other calcium-binding proteins (annexins) in membrane fusion. We have also discussed the roles of other cations, lipid phase transitions, long chain fatty acids and other fusogenic molecules. Finally, we have presented a simple molecular model for the mechanism of lipid membrane fusion, consistent with the experimental evidence and incorporating various elements proposed previously.     

Concentration effects of volatile anesthetics on the properties of model membranes: a coarse-grain approach

To gain insights into the molecular level mechanism of drug action at the membrane site, we have carried out extensive molecular dynamics simulations of a model membrane in the presence of a volatile anesthetic using a coarse-grain model. Six different anesthetic (halothane)/lipid (dimyristoylphosphatidylcholine) ratios have been investigated, going beyond the low doses typical of medical applications. The volatile anesthetics were introduced into a preassembled fully hydrated 512-molecule lipid bilayer and each of the molecular dynamics simulations were carried out at ambient conditions, using the NPT ensemble. The area per lipid increases monotonically with the halothane concentration and the lamellar spacing decreases, whereas the lipid bilayer thickness shows no appreciable differences and only a slight increase upon addition of halothane. The density profiles of the anesthetic molecules display a bimodal distribution along the membrane normal with maxima located close to the lipid-water interface region. We have studied how halothane molecules fluctuate between the two maxima of the bimodal distribution and we observed a different mechanism at low and high anesthetic concentrations. Through the investigation of the reorientational motions of the lipid tails, we found that the anesthetic molecules increase the segmental order of the lipids close to the membrane surface.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

Insights into lipid-protein interactions from computer simulations

Lipid-protein interactions play an important direct role in the function of many membrane proteins. We argue they are key players in membrane structure, modulate membrane proteins in more subtle ways than direct binding, and are important for understanding the mechanism of classes of hydrophobic drugs. By directly comparing membrane proteins from different families in the same, complex lipid mixture, we found a unique lipid environment for every protein. Extending this work, we identified both differences and similarities in the lipid environment of GPCRs, dependent on which family they belong to and in some cases their conformational state, with particular emphasis on the distribution of cholesterol. More recently, we have been studying modes of coupling between protein conformation and local membrane properties using model proteins. In more applied approaches, we have used similar methods to investigate specific hypotheses on interactions of lipid and lipid-like molecules with ion channels. We conclude this perspective with some considerations for future work, including a new more sophisticated coarse-grained force field (Martini 3), an interactive visual exploration framework, and opportunities to improve sampling.     

Stopped-flow study of anesthetic effect on water-transport kinetics through phospholipid membranes. Interfacial versus lipid core ligands

We have compared ligand effects between polar and apolar anesthetic molecules upon water transport across phospholipid membranes by kinetic analysis of the osmotic swelling rate, using a stopped-flow technique. Chloroform and 1-hexanol were used as interfacial ligands, and carbon tetrachloride and n-hexane were used as their counterparts, representing lipid core action. Because anesthetics transform the solid-gel membrane into a liquid-crystalline state, and because phospholipid membranes display an anomaly in permeability at the phase transition, dimyristoylphosphatidylcholine vesicles were studied at temperatures above the main phase transition to avoid this anomaly. All these molecules increased the osmotic swelling rate. However, a significant difference was observed in the activation energy, delta Ep, between polar and apolar molecules; delta Ep was almost unaltered by the addition of polar molecules (chloroform and 1-hexanol), whereas it was decreased by apolar molecules (carbon tetrachloride and n-hexane). The obtained results were analyzed in terms of the dissolution-diffusion mechanism for water permeation across the lipid membrane. It is suggested that polar molecules affect water permeability by altering the partition of water between the membrane interior and water phase, and apolar molecules affect it by altering both the partition and the diffusion of water within the membrane interior.     

Direct electrochemistry of spinach plastocyanin at a lipid bilayer-modified electrode: cyclic voltammetry as a probe of membrane-protein interactions.

The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.     

Models of protein lateral arrangements in lipid bilayer membranes. Application to electron spin resonance studies of cytochrome c oxidase

We consider the situation of integral membrane proteins in a lipid bilayer matrix where the size of the polar group of the protein is important in determining the lateral packing of the proteins. We represent the cross-section of the protein hydrophobic core as a hexagon moving on a lattice, and represent the projection of the polar group onto the plane of the bilayer as a shape, parts of which overlap the hexagon. Lattice sites represent lipid molecules. We calculate the fraction of lipid molecules which are adjacent to the hydrophobic core of at least one protein. We use this data to consider the "motion restricted" spectrum observed in electron spin resonance (ESR) probe studies, and compute the dependence of the "motion restricted" fraction upon protein concentration. The resulting curves can be used to analyse ESR data in order to deduce the size and shape of the proteins' polar segment. We have used the range of models examined to study the dependence upon protein concentration of the particular case of the "motion restricted" spectrum of a spin-labelled lipid freely diffusing or, alternatively, covalently bound to cytochrome c oxidase. We find that our calculations are in accord with a model where approximately 60 lipid molecules can fit around an isolated such protein in both halves of the bilayer, and where the polar segment is substantially anisotropic and extends laterally beyond the limits of the hydrophobic core. The latter is in accord with what is known about the structure of cytochrome c oxidase. We indicate further measurements that should be performed in order to establish more definitively the dependence of the "motion restricted" component upon protein concentration, giving the lipid protein ratios at which they should be performed, and we make predictions concerning the results. Finally we argue for a particular unified way of plotting experimental data.     

Molecular order and dynamics in planar lipid bilayers: effects of unsaturation and sterols

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in macroscopically oriented multilayers of saturated [dimyristoylphosphatidylcholine (DMPC)] and unsaturated [palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dilineoylphosphatidylcholine (DLPC), plant digalactosyldiglyceride (DGDG)] lipids with and without cholesterol. In all the lipid systems studied the order parameter (P2) of TMA-DPH molecules was found to be higher than that for DPH. Considerations of the order parameter (P4), however, indicate that DPH molecules have a heterogeneous distribution in bilayers of unsaturated lipids, with a significant fraction of the molecules lying with their long axes parallel to the bilayer planes. Both the DPH and TMA-DPH molecules exhibit a decrease in the molecular order as well as a decrease in their rates of motion on increasing the unsaturation of the hydrocarbon chains. The addition of cholesterol tends to reverse this effect, with an increase in both the order and dynamics. Bilayers of DOPC, however, exhibit a somewhat different result. It is suggested that the discrepancies between these observations and findings with lipid vesicle systems simply reflect the effects of curvature on the behavior of the probe molecules. The results indicate that the concept of membrane fluidity must be used with great caution.     

Behavior of cholesterol and its effect on head group and chain conformations in lipid bilayers: a molecular dynamics study.

Cholesterol molecules were put into a computer-modeled hydrated bilayer of dimyristoyl phosphatidyl choline molecules, and molecular dynamics simulations were run to characterize the effect of this important molecule on membrane structure and dynamics. The effect was judged by observing differences in order parameters, tilt angles, and the fraction of gauche bonds along the hydrocarbon chains between lipids adjacent to cholesterol molecules and comparing them with those further away. It was observed that cholesterol causes an increase in the fraction of trans dihedrals and motional ordering of chains close to the rigid steroid ring system with a decrease in the kink population. The hydrogen-bonding interactions between cholesterol and lipid molecules were determined from radial distribution calculations and showed the cholesterol hydroxyl groups either solvated by water, or forming hydrogen bond contacts with the oxygens of lipid carbonyl and phosphate groups. The dynamics and conformation of the cholesterol molecules were investigated and it was seen that they had a smaller tilt with respect to the bilayer normal than the lipid chains and furthermore that the hydrocarbon tail of the cholesterol was conformationally flexible.     

Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions.

A method of Monte Carlo calculations has been applied to the problem of fluorescence energy transfer in two dimensions in order to provide a quantitative measure of the effects of nonideal mixing of lipid and protein molecules on the quenching profiles of membrane systems. These numerical techniques permit the formulation of a detailed set of equations that describes in a precise manner the quenching and depolarization properties of planar donor-acceptor distributions as a function of specific spectroscopic and organizational parameters. Because of the exact nature of the present numeric method, these results are used to evaluate critically the validity of previous approximate treatments existing in the literature. This method is also used to examine the effects of excluded volume interactions and distinct lattice structures on the expected transfer efficiencies. As a specific application, representative quenching profiles for protein-lipid mixtures, in which donor groups are covalently linked to the protein molecules and acceptor species are randomly distributed within lipid domains, have been obtained. It is found that the existence of phase-separated protein domains gives rise to a shielding effect that significantly decreases the transfer efficiencies with respect to those expected for an ideal distribution of protein molecules. The results from the present numerical study indicate that the experimental application of fluorescence energy transfer measurements in multicomponent membrane systems can be used to obtain organizational parameters that accurately reflect the lateral distribution of protein and lipid molecules within the bilayer membrane.     

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer.

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.     

Identifying the Interaction of Vancomycin With Novel pH-Responsive Lipids as Antibacterial Biomaterials Via Accelerated Molecular Dynamics and Binding Free Energy Calculations

Nano-drug delivery systems have proven to be an efficient formulation tool to overcome the challenges with current antibiotics therapy and resistance. A series of pH-responsive lipid molecules were designed and synthesized for future liposomal formulation as a nano-drug delivery system for vancomycin at the infection site. The structures of these lipids differ from each other in respect of hydrocarbon tails: Lipid1, 2, 3 and 4 have stearic, oleic, linoleic, and linolenic acid hydrocarbon chains, respectively. The impact of variation in the hydrocarbon chain in the lipid structure on drug encapsulation and release profile, as well as mode of drug interaction, was investigated using molecular modeling analyses. A wide range of computational tools, including accelerated molecular dynamics, normal molecular dynamics, binding free energy calculations and principle component analysis, were applied to provide comprehensive insight into the interaction landscape between vancomycin and the designed lipid molecules. Interestingly, both MM-GBSA and MM-PBSA binding affinity calculations using normal molecular dynamics and accelerated molecular dynamics trajectories showed a very consistent trend, where the order of binding affinity towards vancomycin was lipid4?>?lipid1?>?lipid2?>?lipid3. From both normal molecular dynamics and accelerated molecular dynamics, the interaction of lipid3 with vancomycin is demonstrated to be the weakest (?G_binding?=??2.17 and ?11.57, for normal molecular dynamics and accelerated molecular dynamics, respectively) when compared to other complexes. We believe that the degree of unsaturation of the hydrocarbon chain in the lipid molecules may impact on the overall conformational behavior, interaction mode and encapsulation (wrapping) of the lipid molecules around the vancomycin molecule. This thorough computational analysis prior to the experimental investigation is a valuable approach to guide for predicting the encapsulation ability, drug release and further development of novel liposome-based pH-responsive nano-drug delivery system with refined structural and chemical features of potential lipid molecule for formulation development.     

Biological membranes: the importance of molecular detail

Are lipid interactions with membrane proteins best described in terms of the physical properties of the lipid bilayer or in terms of direct molecular interactions between particular lipid molecules and particular sites on a protein? A molecular interpretation is more challenging because it requires detailed knowledge of the 3D structure of a membrane protein, but recent studies have suggested that a molecular interpretation is necessary. Here, the idea is explored that lipid molecules modify the ways that transmembrane α-helices pack into bundles, by penetrating between the helices and by binding into clefts between the helices, and that these effects on helix packing will modulate the activity of a membrane protein.