I. Structural studies on rhodopsin by photoaffinity labeling. II. Bioorganic studies on a rhodopsin analog from a retinal containing a 9-membered ring in the side chain

Photoaffinity labeling of rhodopsin was carried out to identify the amino acids in the binding sites of the retinal and thereby arrive at a possible helical arrangement of the protein. A retinal analog, with a radioactive photolabel (diazoacetate) at position 3 was used for these studies. 14C label 3S-diazoacetoxy-9-cis-retinal bound to bovine opsin and regenerated a chromophore with λmax at 465 nm. Photolysis of the complex at 254 nm resulted in covalent crosslinking of the retinal analog to the protein in 18-20% yield. Proteolytic cleavage (V8 protease) of the crosslinked protein and determination of the distribution of radioactivity indicated that both fragments V8-L (Met1-Glu239) and V8-S (Ser240-Clu341) were labeled. Further cleavage of labeled V8-S with CNBr (in HCOOH) showed that the major crosslinking sites were contained in a 51 residue peptide in helix 6, CNBr c+d (Val258-Met308). An attempt was made to construct a chemical model for bathorhodopsin, the primary photochemical intermediate in the bleaching sequence of rhodopsin