Single-cysteine substitution mutants at amino acid positions 55-75, the sequence connecting the cytoplasmic ends of helices I and II in rhodopsin: Reactivity of the sulfhydryl groups and their derivatives identifies a tertiary structure that changes upon light-activation
J. Klein-seetharaman et al., Single-cysteine substitution mutants at amino acid positions 55-75, the sequence connecting the cytoplasmic ends of helices I and II in rhodopsin: Reactivity of the sulfhydryl groups and their derivatives identifies a tertiary structure that changes upon light-activation, BIOCHEM, 38(25), 1999, pp. 7938-7944
Cysteines were introduced, one at a time, at amino acid positions 55-75 in
the cytoplasmic region connecting helices I and IT in rhodopsin. In each of
the 21 cysteine mutants, the reactive native cysteine residues (C140 and C
316) were replaced by serine. Except for N55C, all mutants formed rhodopsin
like chromophores and had normal photobleaching characteristics. The effici
ency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The
reactivity of the substituted cysteine in each mutant toward 4,4'-dithiodip
yridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and
175C were unreactive to 4-PDS under the conditions used, suggesting that th
ey are embedded in the micelle or protein interior. The mutants V63C, H65C-
T70C,and N73C related rapidly, while the remainder of the mutants reacted m
ore slowly, and varied in reactivity with sequence position. For the mutant
s derivatized with LC-PDS, the rate of release of thiopyridone from the res
ulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investig
ated in the dark and in the Light. Marked changes in the rates of thiopyrid
one release in the Light were found at specific sites. Collectively, the da
ta reveal tertiary interactions of the residues in the sequence investigate
d and demonstrate structural changes due to photoactivation.