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.
Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.
Klein-Seetharaman
J
J
Massachusetts Institute of Technology, Cambridge 02139, USA.
Hwa
J
J
Cai
K
K
Altenbach
C
C
Hubbell
W L
WL
Khorana
H G
HG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry*; D003624:Darkness; D004220:Disulfides / Q000737:chemistry; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D015293:Transducin / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D011725:Pyridines; D013439:Sulfhydryl Reagents; C007542:4,4'-dipyridyl disulfide; C042861:4-thiopyridine; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine