The site of attachment of retinal in bacteriorhodopsin. The epsilon-amino group in Lys-41 is not required for proton translocation.

Abstract

Chymotryptic fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71) of bacteriorhodopsin have been shown previously to reassociate so as to regenerate the native bacteriorhodopsin chromophore in lipid/detergent mixtures and to form functional proton-translocating vesicles.The fragment C-2 has now been selectively methylated with formaldehyde and sodium cyanoborohydride to give the edimethylamino derivatives of Lys-30, 40, and 41 in 96-99% average yield.The methylated and unmethylated C-2 fragments were identical in their ability to reassociate with fragment C-1 and retinal to regenerate the bacteriorhodopsin chromophore and to form functional proton-translocating vesicles.In contrast, dimethylation of the lysine residues of the C-1 fragment gave a derivative which did not form an active complex with unmethylated C-2.We conclude that the +amino group in Lys-41 is not required for Schiff's base formation with retinal at any step in the light-driven proton-translocation cycle.Chymotryptic cleavage of BR' forms a large fragment, C-1 (amino acids 72-248), and a small fragment, C-2 (amino acids 1-71).The separated fragments can be recombined under appropriate conditions to generate the native BR chromophore and to form proton-translocating vesicles (1).In the accompanying report (2), the chymotryptic fragments prepared from [~-'~N]lysine-labeled and unlabeled BR were reconstituted in different combinations, and the site of attachment of retinal in the recombined complexes was studied by resonance Raman spectroscopy.The results showed that the chromophore is not attached to Lys-41 in either the lightadapted (BR 570) or the bleached (M 412) states of BR.We