Abstract
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. It has been proposed that non-enzymatic membrane-spanning proteins may also accelerate lipid flip-flop in a non-specific manner by introducing bilayer defects, thus easing the translocation of polar lipid head groups across the bilayer by allowing them to move along the protein-membrane interface [BBA, 1978, 509, 537; Biophys J, 1981, 33, 373; Biochem, 1994, 33, 6721; Biochem, 2001, 40, 10500]. We examined this possibility using deuterated phospholipids and the gramicidin channels, which have well-defined structure and function that make them ideal candidates for probing protein-membrane interactions. 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 concentration-dependent manner.
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