Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology |
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Authors: | H Sigrist P R Allegrini K Stauffer J Schaller N G Abdulaev E E Rickli P Zahler |
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Affiliation: | 1. Institute of Biochemistry, University of Berne Berne, Switzerland;2. Shemyakin Institute of Bioorganic Chemistry Academy of Sciences, Moscow, U.S.S.R. |
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Abstract: | 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'-dimethylamino-azobenzene-4'-isothiocyanate results in covalent modification of a unique lysine epsilon-amino group of bacteriorhodopsin. Lysine residue 41, located in the amino-terminal chymotryptic fragment, has been identified as the arylisothiocyanate 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. |
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Keywords: | Author to whom all correspondence should be addressed. |
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