Structural features of the C-terminal domain of bovine rhodopsin: a site-directed spin-labeling study.
Eleven single-cysteine substitution mutants have been prepared in the sequence 325-340 of rhodopsin, corresponding to the C-terminal domain. Each of the cysteine mutants was modified with a selective nitroxide reagent to introduce a spin-labeled side chain. The electron paramagnetic resonance spectra of the labeled proteins were analyzed in terms of side chain dynamics. At all sites, the spectra reflected the presence of two populations of different mobility, although one was always dominant. The mobility of the dominant population increased in a regular fashion from the palmitoylation sites at 322C and 323C to the C-terminus, where the spectra resembled those of an unfolded protein. This apparent mobility gradient is only slightly affected in mutants lacking the palmitoyl groups, suggesting that they are not responsible for physically anchoring the C-terminal peptide at one end. Binding of a monoclonal antibody to its epitope at the C-terminus dramatically reduces the mobility of nearby residues, creating a local mobility gradient opposite that in the absence of the antibody. These results indicate that the C-terminal domain of rhodopsin, beyond the palmitoylation sites, is highly disordered and dynamic, resembling an unfolded peptide tethered at one end.
Langen
R
R
Jules Stein Eye Institute, Department of Chemistry and Biochemistry, University of California, Los Angeles 90095, USA.
Cai
K
K
Altenbach
C
C
Khorana
H G
HG
Hubbell
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics; D002417:Cattle; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013113:Spin Labels
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D013113:Spin Labels; D019308:Palmitic Acid; D012243:Rhodopsin; D003545:Cysteine