title: Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology.
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.
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins; D001665:Binding Sites; D002338:Carotenoids; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D006217:Halobacterium; D017879:Isothiocyanates; D010446:Peptide Fragments / Q000032:analysis; D011522:Protons; D013057:Spectrum Analysis; D013861:Thiocyanates; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives*
chemical_list: D017879:Isothiocyanates; D010446:Peptide Fragments; D011522:Protons; D013861:Thiocyanates; C005441:phenylisothiocyanate; D002338:Carotenoids; D001436:Bacteriorhodopsins; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene; D002918:Chymotrypsin