STRUCTURE AND FUNCTION IN RHODOPSIN - CYSTEINE-65 AND CYSTEINE-316 ARE IN PROXIMITY IN A RHODOPSIN MUTANT AS INDICATED BY DISULFIDE FORMATION AND INTERACTIONS BETWEEN ATTACHED SPIN LABELS

To probe proximity relationships between different amino acids in the interhelical loops in the cytoplasmic domain of rhodopsin, we are usin g a general approach in which two cysteine residues are introduced at different locations. Here we report on the characteristics of one such mutant that contains the naturally occuring cysteine 316 near the cyt oplasmic end of helix G and a second cysteine at position 65 (H65C), n ear the cytoplasmic end of helix A, The mutant protein after expressio n in COS-1 cells and reconstitution with Il-cis-retinal can be bound t o anti-rhodopsin antibody ID4-Sepharose at pH 6 in a form that contain s the two cysteines in the free sulfhydryl form. In this form, the mut ant protein reacts as expected with N-ethylmaleimide in the dark at ro om temperature and can be derivatized with nitroxide spin labels. Howe ver, under appropriate conditions, the mutant can be isolated with the cysteines in the disulfide form, which has been characterized by anal ysis of fragments produced on proteolysis with thermolysin. A study of the interactions between nitroxide spin labels attached to the two cy steine residues in the mutant protein indicates that in the dark state they are within about 10 Angstrom of each other. On illumination the distance between the spin labels increases, Collectively, the above re sults show that, upon folding of the mutant opsin in vivo, cysteines 6 5 and 316, and by inference, helices A and G, are in proximal location s and move further apart upon photoactivation.