title: Photoaffinity labeling of bacteriorhodopsin.
abstract: 14C-Labeled optically pure 3S- and 3R-(diazoacetoxy)-all-trans-retinals were incorporated separately into bacterioopsin to reconstitute functional bacteriorhodopsin (bR) analogues, 3S- and 3R-diazo-bRs. UV irradiation at 254 nm generated highly reactive carbenes, which cross-linked the radiolabeled retinals to amino acid residues in the vicinity of the beta-ionine ring. The 3S- and 3R-diazo analogues were found to cross-link, respectively, to cyanogen bromide fragments CN 7/CN9 and CN 8/CN 9. More specifically, Thr121 and Gly122 in fragment CN 7 were found to be cross-linked to the 3S-diazo analogue. The identification of cross-linked residues and fragments favors assignments of the seven helices A-G-F-E-D-C-B or B-C-D-E-F-G-A to helices 1-2-3-4-5-6-7 in the two-dimensional electron density map (Henderson et al., 1975, 1986; Mogi et al., 1987). The present results show that the chromophore chain is oriented with the ionone ring inclined toward the outside of the membrane (the 9-methyl group also faces the extracellular side of the membrane).
authors.affiliation: Department of Chemistry, Columbia University, New York, New York 10027.
authors.forename: W D
authors.initials: WD
authors.lastname: Ding
authors.forename: A
authors.initials: A
authors.lastname: Tsipouras
authors.forename: H
authors.initials: H
authors.lastname: Ok
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: M A
authors.initials: MA
authors.lastname: Gawinowicz
authors.forename: K
authors.initials: K
authors.lastname: Nakanishi
mesh_terms: D000345:Affinity Labels; D001436:Bacteriorhodopsins; D001665:Binding Sites; D010446:Peptide Fragments / Q000302:isolation & purification; D010777:Photochemistry; D011487:Protein Conformation
chemical_list: D000345:Affinity Labels; D010446:Peptide Fragments; D001436:Bacteriorhodopsins