STERIC CONSTRAINTS IN THE RETINAL BINDING POCKET OF SENSORY RHODOPSIN-I

Steric constraints in the retinal binding pocket of sensory rhodopsin I (SR-I) are analyzed by studying effects of sample temperature and retinal analogs. The flash-induced yield of the earliest detected intermediate S610, which corresponds to the K intermediate in the bacteriorhodopsin (BR) photocycle, decreases below 220 K and reaches zero at 100 K, while K formation is independent of temperature. The reduced S610 formation at low temperatures indicates a more restricted retinal binding pocket in SR-I during primary photochemical events. Introduction of bulky substituents on the retinal polyene chain in four retinal analogs greatly retards or blocks the final step of chromophore binding to the apoprotein of SR-I. Except for the 14-methyl substitution, these modifications exhibit little or no effect on chromophore binding to BR apoprotein. These results corroborate that the retinal polyene chain binding domain in SR-I is more sterically constrained than that of the retinal pocket in BR. Deletion of the beta-ionone ring renders the analog SR-I pigments nonfunctional, as does deletion of the 13-methyl group, but the corresponding BR analogs are both photochemically and physiologically active. In contrast to the corresponding BR analog, photolysis of the analog SR-I reconstituted with 13-desmethylretinal does not produce an S610-like intermediate at room temperature. The above results and the previous findings that protein constraints inhibit the accommodation of a stable 13-cis-retinal configuration in SR-I suggest a model in which the 13-methyl group functions as a fulcrum to permit movement of one or both ends of retinal to overcome an energy barrier against isomerization.