Reassembly of protein-lipid complexes into large bilayer vesicles: Perspectives for membrane reconstitution

Abstract

Protein-lipid complexes in apolar solvents reassemble into large bilayer protein-lipid vesicles (PLV) with diameters of several micrometers. PLV form spontaneously upon hydration of the protein-lipid complex residue after solvent removal. This procedure was applied to the following membrane proteins: bovine and squid rhodopsin, reaction centers from Rhodopseudomonas sphaeroides, beef heart cytochrome c oxidase and acetylcholine receptors from Torpedo californica. PLV have a large internal aqueous space (e.g., 790 .mu.l/mg of lipid for cattle rhodopsin vesicles). Freeze-fracture replicas of PLV revealed that both internal and external leaflets contained numerous intramembranous particles with diameters between 80-120 .ANG., depending on the specific protein incorporated in the membrane. The optical spectral properties of rhodopsin and reaction centers in PLV were similar to those recorded in the respective natural membrane. Bovine rhodopsin in PLV was chemically regenerable with 9-cis-retinal. Actinic illumination induced proton efflux from reaction center vesicles that was abolished by proton ionophores. Therefore, this method is suitable for the incorporation of some membrane proteins in their functional state. PLV were penetrated with microelectrodes and visualized by the injection of a fluorescent dye. Preliminary electrical recordings were obtained by sealing PLV to a hole in a septum separating 2 aqueous compartments. PLV assembled by this procedure permit the simultaneous analysis of reconstituted membranes by chemical, optical and electrical techniques.