Retinal migration during dark reduction of bacteriorhodopsin.

Abstract

When the retinal Schiff base in chymotryptically cleaved bacteriorhodopsin is reduced to a secondary retinylamine by prolonged exposure to 10% (wt/vol) sodium cyanoborohydride, at pH 10, in the absence of light, approximately 45% of the retinal is found linked to Lys-41 and 22% to Lys-40, and the remainder is scattered over various sites on the large chymotryptic fragment, including the physiological site at Lys-216. The retinal-binding site is destroyed or blocked by the reduction conditions, but the bacteriorhodopsin lattice remains intact. The results demonstrate that artifactual linkage to Lys-40/41 is possible under special conditions. Under these conditions, the epsilon-amino groups of Lys-40/41 show an enhanced ability to form retinylidene linkages with the retinal released by the physiological linkage site at Lys-216, due to some combination of close proximity to the normal linkage site, and increased reactivity with respect to other lysine epsilon-amino groups. The results are of interest for the characterization of the two newly discovered rhodopsin-like proteins, halorhodopsin and slow rhodopsin.