Mapping light-dependent structural changes in the cytoplasmic loop connecting helices C and D in rhodopsin: a site-directed spin labeling study.
All 20 single cysteine substitution mutants in the sequence Y136-M155 of bovine rhodopsin have been prepared and modified with a sulfhydryl-specific nitroxide reagent. This sequence contains the C-D interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra were analyzed, both in the dark and after photoexcitation. Accessibility data show that the rhodopsin polypeptide crosses an aqueous/hydrophobic boundary near V138 and H152. The nitroxide mobilities inferred from the spectra are consistent with a model where the C helix extends to at least residue C140, with much of the helix surface in contact with protein rather than lipid near the cytoplasmic surface of the membrane. Upon photoexcitation, electron paramagnetic resonance spectral changes are observed at sites on the putative C helix surface that are in contact with the protein and at specific sites in the C-D interhelical loop. A simple interpretation of these results is that photoexcitation involves a rigid body movement of the C helix relative to the others in the helix bundle.
Farahbakhsh
Z T
ZT
Jules Stein Eye Institute, Los Angeles, California, USA.
Ridge
K D
KD
Khorana
H G
HG
Hubbell
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003593:Cytoplasm / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D013113:Spin Labels
D013113:Spin Labels; D012243:Rhodopsin