title: A new method for the reconstitution of membrane proteins into giant unilamellar vesicles.
abstract: In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.
authors.affiliation: Laboratoire [corrected] Physico Chimie Cioue, Unité Mixte de Recherche 168 Centre National de la Recherche Scientifique/Institut Curie, 75231 Paris Cedex 05, France. philippe.girard@curie.fr
authors.forename: Philippe
authors.initials: P
authors.lastname: Girard
authors.forename: Jacques
authors.initials: J
authors.lastname: Pécréaux
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lenoir
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Rigaud
authors.forename: Patricia
authors.initials: P
authors.lastname: Bassereau
mesh_terms: D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry*; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D011510:Proteolipids / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D001436:Bacteriorhodopsins; D000252:Calcium-Transporting ATPases