Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: a site-directed spin-labeling study.
Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of arc on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI (Altenbach, C., Yang, K., Farrens, D., Farahbakhsh, Z., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470).
Altenbach
C
C
Jules Stein Eye Institute, Department of Chemistry and Biochemistry, University of California, Los Angeles 90095-7008, USA.
Klein-Seetharaman
J
J
Hwa
J
J
Khorana
H G
HG
Hubbell
W L
WL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003497:Cyclic N-Oxides / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008027:Light; D008823:Micelles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics; D013113:Spin Labels; D013439:Sulfhydryl Reagents / Q000737:chemistry
D003497:Cyclic N-Oxides; D008823:Micelles; D010446:Peptide Fragments; D013113:Spin Labels; D013439:Sulfhydryl Reagents; C026058:EDDA; C013110:metarhodopsins; D012243:Rhodopsin; D004492:Edetic Acid