13] Gentle and fast transmembrane reconstitution of membrane proteins

Abstract

This chapter discusses the gentle and fast transmembrane reconstitution of membrane proteins. A technique is developed for gentle and rapid functional reconstitution of integral membrane proteins into lipid vesicles based on the observation that unilamellar lipid vesicles form spontaneously after mixing aqueous suspensions of long-chain lecithins with small amounts of micellar synthetic short-chain lecithins. The protocol for spontaneous insertion of the light-energized H + -pump baeteriorhodopsin (BR) into lipid vesicles composed of various long-chain phospholipids by aid of the short-chain phospholipid diheptanoylphosphatidylcholine is described in the chapter. BR has been selected as model membrane protein because it is one of the best characterized transmembrane proteins, and therefore has been used also in several other reconstitution procedures. Furthermore, the purple membrane applied as starting material can be easily isolated in pure form without the use of detergents and contains, in addition to BR, only about seven native lipids per protein. The novel method presented allows rapid, easy, and gentle functional reconstitution of other membrane systems and isolated membrane proteins as well.