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 β-ionone ring. The 3S- and 3R-diazo analogues were found to cross-link, respectively, to cyanogen bromide fragments CN 7/CN 9 and CN 8/CN 9. More specifically, Thrl21 and Gly 122 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). © 1990 American Chemical Society. All rights reserved.