Structure and function in rhodopsin. Cysteines 65 and 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 using 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 occurring cysteine 316 near the cytoplasmic end of helix G and a second cysteine at position 65 (H65C), near the cytoplasmic end of helix A. The mutant protein after expression in COS-1 cells and reconstitution with 11-cis-retinal can be bound to anti-rhodopsin antibody 1D4-Sepharose at pH 6 in a form that contains the two cysteines in the free sulfhydryl form. In this form, the mutant protein reacts as expected with N-ethylmaleimide in the dark at room temperature and can be derivatized with nitroxide spin labels. However, under appropriate conditions, the mutant can be isolated with the cysteines in the disulfide form, which has been characterized by analysis of fragments produced on proteolysis with thermolysin. A study of the interactions between nitroxide spin labels attached to the two cysteine residues in the mutant protein indicates that in the dark state they are within about 10 A of each other. On illumination the distance between the spin labels increases. Collectively, the above results show that, upon folding of the mutant opsin in vivo, cysteines 65 and 316, and by inference, helices A and G, are in proximal locations and move further apart upon photoactivation.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
K
K
Yang
D L
DL
Farrens
C
C
Altenbach
Z T
ZT
Farahbakhsh
W L
WL
Hubbell
H G
HG
Khorana
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013820:Thermolysin / Q000737:chemistry
D004220:Disulfides; D008565:Membrane Proteins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D012243:Rhodopsin; D013820:Thermolysin; D003545:Cysteine