PROBING OF THE RETINAL BINDING-SITE OF BACTERIORHODOPSIN BY AFFINITY LABELING

The position of the chromophore within bacteriorhodopsin has been iden tified by cross-linking a cysteine group, introduced by site-specific mutagenesis, with a chromophore suitably derivatized with an active le aving group. Since bacteriorhodopsin has no cysteines, a site-specific cysteine mutant will contain only one free sulfhydryl group capable o f reacting with the retinal analog. Met118, Thr121, and Ser141 were se lected to be mutated to cysteine. No pigment absorbing in the visible region was obtained for the Ser141Cys mutant. The Met118Cys and Thr121 Cys mutants have similar absorption maxima, proton pumping efficiencie s and photocycles to those of the wild-type pigment. 4-Bromoretinal, i n which the reactive allylic halide readily undergoes nucleophilic dis placement, was used as the reactive chromophore. Pigments were obtaine d on reaction of all-trans-4-bromoretinal with the apoproteins of Met1 18Cys, Thr121Cys, and wild-type bacteriorhodopsin (lambda(max) = 464-4 70 nm). Analysis of the denatured pigments on SDS-polyacrylamide gels showed incorporation of tritiated chromophore into the Met118Cys mutan t but not into the wild-type or Thr121Cys pigments. Met118Cys apoprote in which was preincubated with the cysteine-specific reagent N-ethylma leimide formed a pigment with 4-bromoretinal but no cross-linking was observed, providing evidence that the cross-linking of the chromophore is to the cysteine at 118. We conclude that Met118 is positioned in t he chromophore binding pocket, proximal to the C-4 position of cyclohe xyl ring of retinal.