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A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

Philippe Girard ^*, Jacques Pécréaux ^*, Guillaume Lenoir , Pierre Falson , Jean-Louis Rigaud ^*  and Patricia Bassereau ^*

^* Laboratorie Physico Chimie Cioue, Unité Mixte de Recherche 168 Centre National de la Recherche Scientifique/Institut Curie, 75231 Paris Cedex 05, France; and Laboratoire de Recherche Correspondant 34V Commissariat à l'Energie Atomique, Institut Curie, 75231 Paris Cedex 05, France; Commissariat à l'Énergie Atomique, Centre d'Etudes de Saclay, Direction des Sciences du Vivant, Département de Biologie Joliot Curie, Service de Biophysique des Fonctions Membranaires, Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique, 91191 Gif-sur-Yvette Cedex, France

Correspondence: Address reprint requests to Philippe Girard, E-mail: philippe.girard{at}curie.fr.

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 Ca²⁺-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 µm 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 Ca²⁺ pumping driven by bacteriorhodopsin or Ca²⁺-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 µm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.

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