Unlike transmembrane proteins, phospholipids can reorient within the membrane by moving from one leaflet to the other. Spontaneous lipid translocation tends to be very slow, however, and cells facilitate the process with enzymes that catalyze the transmembrane lipid movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a non-specific 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 function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. Making use of recently developed protocol and assays for the preparation and characterization of asymmetric lipid vesicles, we studied compositionally and isotopically asymmetric proteoliposomes containing gramicidin. Protein incorporation, conformation and function were examined with small-angle x-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay. Differential scanning calorimetry revealed the effect of the protein on the melting transition temperatures of the two bilayer leaflets, which over time merged into a single peak indicating lipid scrambling. Using proton NMR, we monitored the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles with and without the protein. 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 amount of membrane deformation in the samples. 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.

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

2019 Spring National Meeting

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