Regeneration of the native bacteriorhodopsin structure from two chymotryptic fragments.

Abstract

Chymotrypsin cleaves bacteriorhodopsin to two fragments: C-1, amino acids 72-248, and C-2, amino acids 1-71. Denaturation and renaturation of these fragments have been studied. Following denaturation in sodium dodecyl sulfate, both C-1 and C-2 regain secondary structure in phospholipid/cholate/sodium dodecyl sulfate mixed micelles. When combined, they form a complex with a secondary structure resembling that of bacterio-opsin. However, on further incubation in phospholipid/cholate/sodium dodecyl sulfate, separate fragments as well as the C-1 and C-2 complex denature again. Retinal binds tightly to the C-1 and C-2 complex (Kb greater than 10(7) M-1) and stabilizes the folded conformation. The formation of the complex of C-1, C-2, and retinal is maximal at pH 6.0. The ternary complex contains two species: one which absorbs similarly to the light-adapted purple membrane and a second with a lambda max between 450 and 500 nm. The formation of the latter species is favored at higher temperatures and is reversible. Vesicles formed from the ternary complex of C-1, C-2, and retinal translocate protons at a level close to that of intact bacteriorhodopsin.