Synthesis of a photoaffinity-labeled (11Z)-retinal: Identification of retinal/rhodopsin cross-linked sites along the visual-transduction path

The retinal chromophore (11Z)-3-diazo-4-oxoretinal (1) with two photo-labil e moieties has been synthesized by semi-hydrogenation of an 11-yne precurso r with activated Zn in aqueous media. Incorporation of 1 into opsin yielded diazoketo rhodopsin (DK-Rh), which, upon bleaching, gave rise to intermedi ates batho-Rh, lumi-Rh, meta-Rh, and meta-II-Rh corresponding to those of n ative Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and the se bleaching intermediates showed that the ionone ring cross-linked to Trp2 65 of helix F in DK-Rh and bathe intermediate, and to Ala169 of helix D in lumi, meta-I, and meta-II intermediates. These results demonstrate the occu rrence of large conformational changes along the visual transduction path, which, in turn, is responsible for activation of the G-protein.