29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

The effect of steric hindrance between retinal and protein on the function of rhodopsin

Abstract-
A series of retinal analogues has been synthesized with altered intramolecular (10-methyl-, 10-methyl-13-demethyl-, and 13-demethyl retinal) and intermolecular (9-demethyl-, 9-ethyl, and 9-isopropyl retinal) steric hindrance. These chromophore derivatives were incorporated into bovine opsin, and the thus formed visual pigments were characterized for: (i) their kinetics of pigment formation, (ii) their stability towards hydroxyl amine in the dark, (iii) their bleaching quantum yield, and (iv) their bleaching kinetics in the microseconds-to-seconds time range upon light exposure (laser-induced flash photolysis). All visual pigment derivatives showed absorption maxima similar as the native pigment between 490 and 510 nm with the exception of the 9-demethyl derivative, which absorbed significantly hypsochromic at 460 nm. The pigment formation kinetics revealed an optimized fit for the native chromophore, retinal. Both, 9- and 13-demethyl retinal reconstituted slower, probably due to increased conformational flexibility within the binding pocket. The compounds with enlarged or alternately positioned substituents were incorporated slower. The most hindered compound, 9-isopropyl retinal, yielded only ca. 3% of assembled rhodopsin. The bleaching quantum yield for the intramolecular-hindered analogs (10-methyl-, 10-methyl-13-demethyl-, and 13-demethyl retinal) also showed the influence of the altered substitution pattern, whereas 9-demethyl and 9-ethyl retinal revealed a nearly identical bleaching behavior as the native pigment. A time-resolved analysis of the bleaching process demonstrated the strongest effects of the modified substitution pattern: Whereas 9-demethyl retinal caused an acceleration of the bleaching kinetics, most drastic in the formation of the meta-II intermediate, all other retinal derivatives showed a much slower and also altered formation of the meta-(II) intermediate. In the final bleaching reaction, 10-methyl-13-demethyl retinal exhibited the slowest kinetics of all analog pigments with a time constant of 10 hours. We wish to thank Prof. S.E. Braslavsky, MPI Muelheim, for providing the flash photolysis equipment. A.O. is a recipient of a grant from the Friedrich-Ebert-Foundation.

Keywords: rhodopsin, retinal analogs, chromophore-protein interaction, flash photolysis