THE LOCATION OF THE CHROMOPHORE IN RHODOPSIN - A PHOTOAFFINITY STUDY

A photoreactive analog of 11-cis-retinal was synthesized and used in p hoto-cross-linking studies to determine the orientation of the chromop hore in bovine rhodopsin. The photoaffinity analog incorporated a trit ium in the aldehyde group, a photoactivable diazo ketone moiety on the beta-ionone ring, and a six-membered ring in the side chain; this six -membered ring fixes the 11-ene in its cisoid form, thus preventing ph otoisomerization and scrambling of cross-linked sites, The retinal ana log was incorporated into bovine opsin to yield a rhodopsin analog abs orbing maximmally at 483 nm. UV irradiation of the diazo ketone functi on resulted in almost exclusive cross-linking to Trp-265 and Leu-266 i n alpha-helix F. These amino acids were identified by Edman degradatio n of a cyanogen bromide-cleaved peptide, which was separated by HPLC a nd gel electrophoresis. Since the labeled amino acids are located in t he middle of the transmembrane helix F, while the Schiff-base linkage (Lys-296) at the other terminus of the chromophore also resides in the middle of helix G, the entire chromophore is positioned near the cent er of the lipid bilayer. A previous study (Nakayama, T. A.; Khorana, H . G. J. Biol. Chem. 1990, 265, 15762-15769) using a photoisomerizable and photoactive 11-cis-retinal analog demonstrated that both helices C and F were cross-linked. The present exclusive labeling of helix F wi th the nonisomerizable analog indicates that C-3 of the beta-ionone ri ng is in contact with helix F in rhodopsin, and that light-induced iso merization moves the C-3 region to come in contact with helix C. This motion could be responsible for triggering the conformational changes leading to metarhodopsin-II, the species directly involved in initiati ng the enzymatic cascade leading to visual transduction.