Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: Proximities between amino acids deduced from spontaneous disulfide bond formation between cysteine pairs engineered in cytoplasmic loops 1, 3,and 4

To probe proximities between amino acids in the cytoplasmic domain by using mutants containing engineered cysteine pairs, three sets of rhodopsin muta nts have been prepared. In the first two sets, a cysteine was placed, one a t a time, at positions 311-314 in helix VIII, while the second cysteine was fixed at position 246 (set I) and at position 250 (set II) at the cytoplas mic end of helix VI. In the third set, one cysteine was fixed at position 6 5 while the second cysteine was varied between amino acid positions 306 and 321 located at the cytoplasmic end of helix VII and throughout in helix VI II. Rapid disulfide bond formation in the dark was found between the cystei ne pairs in mutants A246C/Q312C, -A246C/K311C I C and in mutants H65C/C316, H65C/315C and H65C/312C. Disulfide bond formation at much lower rates was found in mutants A246C/F313C, V250C/Q312C, H65C/N310C, H65C/K311C, H65C/F31 3C, and H65C/R314C; the remaining mutants showed no significant disulfide b ond formation. Comparisons of the results from disulfide bond formation in solution with the distances observed in the rhodopsin crystal structure sho wed that the rates of disulfide bond formation in most cases were consisten t with the amino acid proximities as revealed in crystal structure. However , deviations were also found, in particular, in the set containing fixed cy steine at position Cys246 and cysteines at positions 311-314. The results i mplicate significant effects of structural dynamics on disulfide bond forma tion in solution.