Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid.
The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.
D000818:Animals; D001436:Bacteriorhodopsins / Q000378:metabolism* / Q000648:ultrastructure; D004134:Dimyristoylphosphatidylcholine; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006217:Halobacterium / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers; D008099:Liver / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines; D012542:Scattering, Radiation; D013552:Swine; D013816:Thermodynamics
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D001436:Bacteriorhodopsins; D014453:Glucuronosyltransferase; C017251:1,2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine