Delipidation, Renaturation, and Reconstitution of Bacteriorhodopsin

This chapter describes the complete displacement of the purple membrane lipids by detergents and the subsequent reconstitution of the solubilized bacteriorhodopsin with exogenous or endogenous lipids to form functional vesicles. In these vesicles, all protein molecules are oriented with their COOH-termini exposed at the external surface. The opposite orientation is found in the intact cell membrane of Halobacterium halobium. In addition, a method is given for the complete denaturation by organic solvents of lipid- and detergent-free bacterioopsin. The denatured protein can be refolded and becomes fully active when reconstituted into vesicles. The effects of different lipids and retinal analogs on proton translocating activity can be tested using vesicles made by the reconstitution procedures. The vesicles formed from the endogenous lipids have extraordinary properties, including high stability, optical clarity, and such high impermeability to ions that extensive proton translocation into the vesicles is observed only in the presence of K⁺ and valinomycin, which together prevent formation of a membrane potential. The studies of denaturation and renaturation bear on the problem of membrane biogenesis.