Photoaffinity labeling of bacteriorhodopsin.
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).
Ding
W D
WD
Department of Chemistry, Columbia University, New York, New York 10027.
Tsipouras
A
A
Ok
H
H
Yamamoto
T
T
Gawinowicz
M A
MA
Nakanishi
K
K
D000345:Affinity Labels; D001436:Bacteriorhodopsins; D001665:Binding Sites; D010446:Peptide Fragments / Q000302:isolation & purification; D010777:Photochemistry; D011487:Protein Conformation
D000345:Affinity Labels; D010446:Peptide Fragments; D001436:Bacteriorhodopsins