Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol

AbstractWe report the first 4-component phase diagram for the lipid bilayer mixture, DSPC/DOPC/POPC/chol (distearoylphosphatidylcholine/dioleoylphosphatidylcholine/1-palmitoyl, 2-oleoylphosphatidylcholine/cholesterol). This phase diagram, which has macroscopic Ld+Lo phase domains, clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has nanoscopic phase domains of Ld+Lo. Our studies start from two published ternary phase diagrams, and show how these can be combined into a quaternary phase diagram by study of a few hundred samples of intermediate compositions

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

© 2019 Biophysical Society Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes

Nuclear Magnetic Relaxation Studies of Lecithin Bilayers

In an effort to elucidate the state of molecular motion in aqueous lecithin systems, the nuclear magnetic relaxation properties of unsonicated lecithin crystal and liquid crystal phases have been investigated. The presence of a narrow component in the nmr spectrum of aqueous lamellar lecithin crystals is taken to indicate a significant degree of motion of the lecithin methyl groups in this solid phase. Also in the crystal state of lecithin, nmr linewidth measurements, together with thermal analysis experiments appear to indicate that a structural rearrangement of either the lecithin or the bound water takes place, which is not accompanied by an increase in molecular mobility. Delayed Fourier Transform and wide-line nmr experiments show that at the crystal -> liquid crystal (bilayer) phase transition temperature, the lecithin choline methyl, terminal methyl, and hydrocarbon chain methylene protons are all simultaneously mobilized. A previously noted field dependence of the proton magnetic resonance linewidth of aqueous lecithin bilayers is shown to be accounted for by the chemical shift differences among the various kinds of protons. Spin lattice relaxation rates have been measured for these protons as a function of temperature and frequency, and these data have been interpreted in terms of models for the segmental motion of the choline head groups and the hydrocarbon chains. The influence of spin diffusion on the relaxation behavior of the various protons is also discussed