TY - JOUR
T1 - Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin
T2 - A site-directed spin-labeling study
AU - Altenbach, Christian
AU - Yang, Ke
AU - Farrens, David L.
AU - Farahbakhsh, Zohreh T.
AU - Gobind Khorana, H.
AU - Hubbell, Wayne L.
PY - 1996
Y1 - 1996
N2 - Thirty consecutive single cysteine substitution mutants in the amino acids Q225-1256 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 data 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 α- 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.
AB - Thirty consecutive single cysteine substitution mutants in the amino acids Q225-1256 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 data 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 α- 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.
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U2 - 10.1021/bi960849l
DO - 10.1021/bi960849l
M3 - Article
C2 - 8823182
AN - SCOPUS:0029818551
SN - 0006-2960
VL - 35
SP - 12470
EP - 12478
JO - Biochemistry
JF - Biochemistry
IS - 38
ER -