Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study.
Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin have been prepared and modified with a sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with hydrophilic and hydrophobic paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra analyzed in terms of side chain mobility, both in the dark and after photoactivation. Accessibility cata shows that the rhodopsin polypeptide chain crosses an aqueous/ hydrophobic boundary in the range V227-K231 and again in the range V250-V254. In the hydrophobic segments, both the accessibility and mobility data are consistent with helical structures. In the regions of the sequence located within the aqueous phase, periodic variation in both accessibility and mobility of the spin-labeled side chains indicates that the E-F interhelical loop is largely alpha-helical, being formed by regular extensions of the E and F helices by about 1.5 and 3 turns, respectively. Judging from nitroxide mobilities, the putative extension of helix E in the aqueous phase is more dynamic than that of helix F. Changes in the electron paramagnetic resonance characteristics of the spin-labeled rhodopsin upon photoactivation indicate that chromophore isomerization results in patterns of structural changes that can be interpreted in terms of movements of helices that extend into the aqueous loop regions.
Altenbach
C
C
Jules Stein Eye Institute, University of California, Los Angeles 90095-7008, USA.
Yang
K
K
Farrens
D L
DL
Farahbakhsh
Z T
ZT
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; D016296:Mutagenesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels / Q000138:chemical synthesis; D013439:Sulfhydryl Reagents
D013113:Spin Labels; D013439:Sulfhydryl Reagents; D012243:Rhodopsin