Atomically detailed lipid bilayer models for the interpretation of small angle neutron and X-ray scattering data

We present a new atom density profile (ADP) model and a statistical approach for extracting structural characteristics of lipid bilayers from X-ray and neutron scattering data. Models for five lipids with varying head and tail chemical composition in the fluid phase, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), are optimized using a simplex based method to simultaneously reproduce both neutron and X-ray scattering data. Structural properties are determined using statistical analysis of multiple optimal model structures. The method and models presented make minimal assumptions regarding the atomic configuration, while taking into account the underlying physical properties of the system. The more general model and statistical approach yield data with well defined uncertainties, indicating the precision in determining density profiles, atomic locations, and bilayer structural characteristics. Resulting bilayer structures include regions exhibiting large conformational variation. Due to the increased detail in the model, the results demonstrate the possibility of a distinct hydration layer within the interfacial (backbone) region.     

Reparameterization of all-atom dipalmitoylphosphatidylcholine lipid parameters enables simulation of fluid bilayers at zero tension

Molecular dynamics simulations of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers using the CHARMM27 force field in the tensionless isothermal-isobaric (NPT) ensemble give highly ordered, gel-like bilayers with an area per lipid of approximately 48 A(2). To obtain fluid (L(alpha)) phase properties of DPPC bilayers represented by the CHARMM energy function in this ensemble, we reparameterized the atomic partial charges in the lipid headgroup and upper parts of the acyl chains. The new charges were determined from the electron structure using both the Mulliken method and the restricted electrostatic potential fitting method. We tested the derived charges in molecular dynamics simulations of a fully hydrated DPPC bilayer. Only the simulation with the new restricted electrostatic potential charges shows significant improvements compared with simulations using the original CHARMM27 force field resulting in an area per lipid of 60.4 +/- 0.1 A(2). Compared to the 48 A(2), the new value of 60.4 A(2) is in fair agreement with the experimental value of 64 A(2). In addition, the simulated order parameter profile and electron density profile are in satisfactory agreement with experimental data. Thus, the biologically more interesting fluid phase of DPPC bilayers can now be simulated in all-atom simulations in the NPT ensemble by employing our modified CHARMM27 force field.     

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca²⁺ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.     

Lipid Organization in Mixed Lipid Membranes Driven by Intrinsic Curvature Difference

Laurdan fluorescence, novel spectral fitting, and dynamic light scattering were combined to determine lateral lipid organization in mixed lipid membranes of the oxidized lipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), and each of the three bilayer lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). Second harmonic spectra were computed to determine the number of elementary emissions present. All mixtures indicated two emissions. Accordingly, spectra were fit to two log-normal distributions. Changes with PGPC mole fraction, X_PGPC, of the area of the shorter wavelength line and of dynamic light scattering-derived aggregate sizes show that: DPPC and PGPC form component-separated mixed vesicles for X_PGPC ≤ 0.2 and coexisting vesicles and micelles for X_PGPC > 0.2 in gel and liquid-ordered phases and for all X_PGPC in the liquid-disordered phase; POPC and PGPC form randomly mixed vesicles for X_PGPC ≤ 0.2 and component-separated mixed vesicles for X_PGPC > 0.2. DOPC and PGPC separate into vesicles and micelles. Component segregation is due to unstable inhomogeneous membrane curvature stemming from lipid-specific intrinsic curvature differences between mixing molecules. PGPC is inverse cone-shaped because its truncated tail with a terminal polar group points into the interface. It is similar to and mixes with POPC, also an inverse cone because of mobility of its unsaturated tail. PGPC is least similar to DOPC because mobilities of both unsaturated tails confer a cone shape to DOPC, and PGPC separates form DOPC. DPPC and PGPC do not mix in the liquid-disordered phase because mobility of both tails in this phase renders DPPC a cone. DPPC is a cylinder in the gel phase and of moderate similarity to PGPC and mixes moderately with PGPC.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Biomembrane simulations of 12 lipid types using the general amber force field in a tensionless ensemble

The AMBER family of force fields is one of the most commonly used alternatives to describe proteins and drug-like molecules in molecular dynamics simulations. However, the absence of a specific set of parameters for lipids has been limiting the widespread application of this force field in biomembrane simulations, including membrane protein simulations and drug-membrane simulations. Here, we report the systematic parameterization of 12 common lipid types consistent with the General Amber Force Field (GAFF), with charge-parameters determined with RESP at the HF/6–31G(d) level of theory, to be consistent with AMBER. The accuracy of the scheme was evaluated by comparing predicted and experimental values for structural lipid properties in MD simulations in an NPT ensemble with explicit solvent in 100:100 bilayer systems. Globally, a consistent agreement with experimental reference data on membrane structures was achieved for some lipid types when using the typical MD conditions normally employed when handling membrane proteins and drug-membrane simulations (a tensionless NPT ensemble, 310?K), without the application of any of the constraints often used in other biomembrane simulations (such as the surface tension and the total simulation box area). The present set of parameters and the universal approach used in the parameterization of all the lipid types described here, as well as the consistency with the AMBER force field family, together with the tensionless NPT ensemble used, opens the door to systematic studies combining lipid components with small drug-like molecules or membrane proteins and show the potential of GAFF in dealing with biomembranes.     

Geometrical effects of phospholipid olefinic bonds on the structure and dynamics of membranes: A molecular dynamics study

The trans isomers of fatty acids are found in human adipose tissue. These isomers have been linked with deleterious health effects (e.g., coronary artery disease). In this study, we performed molecular dynamics simulations to investigate the structures and dynamic properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-elaidoyl sn-glycero-3-phosphatidylcholine (PEPC) lipid bilayers. The geometry of the olefinic bond and membrane packing effects significantly influenced the conformations and dynamics of the two CC single bonds adjacent to the olefinic bond. For the PEPC lipid, the two CC single bonds adjacent to the olefinic bond adopted mainly nonplanar skew–trans and planar cis–trans motifs; although the cis conformation featured relatively strong steric repulsion, it was stabilized through membrane packing because its planar structure is more suitable for membrane packing. Moreover, membrane packing effects stabilized the planar transition state for conformational conversion to a greater extent than they did with the nonplanar transition state, thereby affecting the dynamics of conformational conversion. The rotational motions of the first neighboring CC single bonds were much faster than those of typical saturated CC single bonds; in contrast, the rotational motions of the second neighboring CC single bonds were significantly slower than those of typical saturated torsion angles. The packing of PEPC lipids is superior to that of POPC lipids, leading to a smaller area per lipid, a higher order parameter and a smaller diffusion coefficient. The distinct properties of POPC and PEPC lipids result in PEPC lipids forming microdomains within a POPC matrix.     

Depolarization Laplace Transform Analysis of Exchangeable Hyperpolarized Xe for Detecting Ordering Phases and Cholesterol Content of Biomembrane Models

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon (¹²⁹Xe) atoms in cryptophane-A-monoacid (CrA_ma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrA_ma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75–98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%.     

Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study

Lipid peroxidation plays an important role in cell membrane damage. We investigated the effect of lipid peroxidation on the properties of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) lipid bilayers using molecular dynamics simulations. We focused on four main oxidation products of linoleic acid with either a hydroperoxide or an aldehyde group: 9-trans, cis-hydroperoxide linoleic acid, 13-trans, cis-hydroperoxide linoleic acid, 9-oxo-nonanoic acid, and 12-oxo-9-dodecenoic acid. These oxidized chains replaced the sn-2 linoleate chain. The properties of PLPC lipid bilayers were characterized as a function of the concentration of oxidized lipids, with concentrations from 2.8% to 50% for each oxidation product. The introduction of oxidized functional groups in the lipid tail leads to an important conformational change in the lipids: the oxidized tails bend toward the water phase and the oxygen atoms form hydrogen bonds with water and the polar lipid headgroup. This conformational change leads to an increase in the average area per lipid and, correspondingly, to a decrease of the bilayer thickness and the deuterium order parameters for the lipid tails, especially evident at high concentrations of oxidized lipid. Water defects are observed in the bilayers more frequently as the concentration of the oxidized lipids is increased. The changes in the structural properties of the bilayer and the water permeability are associated with the tendency of the oxidized lipid tails to bend toward the water interface. Our results suggest that one mechanism of cell membrane damage is the increase in membrane permeability due to the presence of oxidized lipids.     

Interactions of Alkylphosphocholines with Model Membranes—The Langmuir Monolayer Study

Alkylphosphocholines (APCs) belong to a class of synthetic antitumor lipids, which are new-generation anticancer agents. In contrast to traditional antitumor drugs, they do not attack the cell nucleus but, rather, the cellular membrane; however, their mechanism of action is not fully understood. This work compared the interactions of selected APCs [namely, hexadecylphosphocholine (miltefosine), octadecylphosphocholine and erucylphosphocholine] with the most important membrane lipids [cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and examined their influence on a model membrane of tumor and normal cells. As a simple model of membranes, Langmuir monolayers prepared by mixing cholesterol either with a saturated phosphatidylcholine (DPPC), for a normal cell membrane, or with an unsaturated one (POPC), for a tumor cell membrane, have been applied. The APC–lipid interactions, based on experimental surface pressure (π) versus mean molecular area (A) isotherms, were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with the ΔG ^exc function). Strong attractive interactions were observed for mixtures of APCs with cholesterol, contrary to the investigated phosphatidylcholines, for which the interactions were found to be weak with a tendency to separation of film components. In ternary monolayers it has been found that the investigated model systems (cholesterol/DPPC/APC vs cholesterol/POPC/APC) differ significantly as regards the interactions between film-forming molecules. The results demonstrate stronger interactions between the components of cholesterol/POPC/APC monolayers compared to cholesterol/POPC film, mimicking tumor cell membranes. In contrast, the interactions in cholesterol/DPPC/APC films were found to be weaker than those in the cholesterol/DPPC system, serving as a model of healthy cell membranes, thus proving that the incorporation of APCs is, from a thermodynamic point of view, unfavorable for binary cholesterol/DPPC monolayers. It can be concluded that the composition of healthy cell membranes is a natural barrier preventing the incorporation of APCs into normal cells.     

Depolarization Laplace Transform Analysis of Exchangeable Hyperpolarized 129Xe for Detecting Ordering Phases and Cholesterol Content of Biomembrane Models

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon (¹²⁹Xe) atoms in cryptophane-A-monoacid (CrA_ma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrA_ma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75–98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%.     

Insertion of Dengue E into lipid bilayers studied by neutron reflectivity and molecular dynamics simulations

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Palmitate incorporation into lipids by lung subcellular fractions

The incorporation of palmitate into lipids by hamster lung subcellular fractions was examined and compared to the simultaneous incorporation of sn-glycero-3-phosphate. The rate of incorporation was greater for the microsomal fraction than for the mitochondria-rich fraction with very little incorporation by the supernatant. The supernatant, however, increased the rate of incorporation by 60–70% when added to the particulate fractions. The presence of CoA, ATP and rac-glycerophosphate in the incubation medium was required for optimal incorporation in all fractions. Comparison of incorporation of sn-glycero-3-phosphate and palmitate into lipids indicated that a great part of palmitate incorporation into 3-sn-phosphatidylcholine did not proceed via the diglyceride pathway. The highest de novo incorporation of palmitate was observed into 3-sn-phosphatidylethanolamine.     

Properties of sonicated vesicles of three synthetic phospholipids

The following synthetic phospholipids were prepared, and the structures that were formed by ultrasonic irradiation in aqueous solution were studied: 1,2-di(10-bromo stearoyl)-3-sn-phosphatidylcholine (DBrPC), 1,2-di(10-methyl stearoyl)-3-sn-phosphatidylcholine (DMePC), and 1-palmitoyl-2-oleyl-3-sn-phosphatidylcholine (POPC). Uniform populations of small, unilamellar vesicles were obtained in all cases by gel filtration on Sepharose 4B. Hydrodynamic and trapped volume measurements show that POPC is nearly identical in size and shape to vesicles of egg phosphatidylcholine whereas DBrPC and DMePC appear to have a non-spherical shape. Fluorescence depolarization measurements show that vesicles from all three lipids are in the liquid crystalline state between 5 and 50°C.The partial specific volume of DMePC is larger than that of egg PC, whereas the partial specific volume of DBrPC is considerably lower; these lipids should therefore be useful in studies requiring the separation of vesicle populations. POPC, being virtually identical in size, shape and bilayer fluidity to egg PC, should be an excellent model of a ‘natural’ lecithin with a defined fatty acid composition.     

Pinched multilamellar structure of aggregates of lysozyme and phosphatidylserine-containing membranes revealed by FRET

Electrostatic interactions between negatively charged membranes and basic peptides/protein domains have been implicated as the driving force for several important processes, often involving membrane aggregation, fusion, or phase separation. Recently, acidic lipids were reported to both catalyze amyloid fiber formation by amyloidogenic proteins/peptides and induce formation of “amyloid-like” fibrils by nonamyloidogenic proteins. This study aims to characterize the structure of the aggregates of a basic protein (lysozyme) and negatively charged membranes (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine 4:1 mixture) at the molecular level, using Förster resonance energy transfer. It is concluded that lysozyme induced formation of a “pinched lamellar” structure, with reduced interbilayer distance in the regions where there is bound protein and increased interbilayer distance (stabilized by hydration repulsion) outside these areas.     

Role of cholesterol flip-flop in oxidized lipid bilayers

We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0–50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol’s flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.     

Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

Universal behavior of membranes with sterols

Lanosterol is the biosynthetic precursor of cholesterol and ergosterol, sterols that predominate in the membranes of mammals and lower eukaryotes, respectively. These three sterols are structurally quite similar, yet their relative effects on membranes have been shown to differ. Here we study the effects of cholesterol, lanosterol, and ergosterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid bilayers at room temperature. Micropipette aspiration is used to determine membrane material properties (area compressibility modulus), and information about lipid chain order (first moments) is obtained from deuterium nuclear magnetic resonance. We compare these results, along with data for membrane-bending rigidity, to explore the relationship between membrane hydrophobic thickness and elastic properties. Together, such diverse approaches demonstrate that membrane properties are affected to different degrees by these structurally distinct sterols, yet nonetheless exhibit universal behavior.