Group-directed modification of bacteriorhodopsin by arylisothiocyanates: Labeling, identification of the binding site and topology

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Group-directed hydrophobic modification of membrane-integrated protein segments by arylisothiocyanates is applied to bacteriorhodopsin. Labeling of purple membrane with phenylisothiocyanate and 4- N,N′-dimethyl-amino-azobenzene-4′-isothiocyanate results in covalent modification of a unique lysine ε-amino group of bacteriorhodopsin. Lysine residue 41, located in the amino-terminal chymotryptic fragment, has been identified as the arylisothio-cyanate binding site by established sequencing techniques. The phenylisothiocyanate binding site is not accessible for the aqueously soluble analog p-sulfophenylisothiocyanate. Furthermore, the acid-induced bathochromic shift of the bound chromophore reagent is not observed following acidification of 4- N,N′-dimethylamino-azobenzene-4′-isothiocyanate-labeled purple membrane. The modification thus occurs in the hydrophobic membrane domain, providing further evidence for intramembraneous disposition of the modified protein segment. Light-induced proton translocation is preserved in reconstituted vesicles containing either phenylisothiocyanate-modified or 4- N,N′-dimethylamino-azobenzene-4′-isothiocyanate-modified bacteriorhodopsin.