Rhodopsin in reconstituted phospholipid vesicles. 1. Structural parameters and light-induced conformational changes...

The structure of purified rhodopsin was investigated by steady-state resonance energy transfer and fluorescence quenching techniques: (1) Fluorescence parameters and relative distances between rhodopsin sites labeled with fluorescent probes and the endogenous chromophore 11-cis-retinal were measured in micellar detergent solution and in reconstituted phospholipid vesicles. (2) The accessibility of the labeled rhodopsin sites in reconstituted vesicles to N-methyl- and N-benzylpicolinium was studied in the dark and subsequent to rhodopsin bleaching. Fluorescent-labeled rhodopsin was affinity purified in octyl glucoside from rod outer segments which were previously reacted with either the sulfhydryl-specific reagents, pyrenylmaleimide or monobromobimane, or reagents specific to amino groups, dansyl chloride or fluorescein isothiocyanate. The purified protein was recombined with phospholipids, and vesicles were formed by detergent dialysis. All four fluorophores appear to react greater than or equal to 30 A away from the endogenous chromophore as estimated from the efficiency of energy transfer and presumably probe rhodopsin domains exposed at the membrane surface. The maximal fraction of quenchable fluorescence and the mean quenching constant were determined in dark and bleached vesicles: bleaching did not affect the quenching of the fluorophores attached to sulfhydryl groups but markedly decreased the quenching constants of the fluorophores coupled to amino groups. The apparent collisional rate constant decreased by 20- and 4-fold for dansyl and fluorescein, respectively. The results suggest that bleaching reduced the accessibility of these sites which, in turn, may reflect light-induced displacements of rhodopsin domains at the membrane surface. Such structural changes may regulate rhodopsin-rhodopsin as well as rhodopsin-enzyme interactions.