Zt. Farahbakhsh et al., MAPPING LIGHT-DEPENDENT STRUCTURAL-CHANGES IN THE CYTOPLASMIC LOOP CONNECTING HELIX-C AND HELIX-D IN RHODOPSIN - A SITE-DIRECTED SPIN-LABELING STUDY, Biochemistry, 34(27), 1995, pp. 8812-8819
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 interhelica
l loop, a transducin interaction site, The accessibilities of the atta
ched nitroxides to collisions with paramagnetic probes in solution wer
e determined, and the electron paramagnetic resonance spectra were ana
lyzed, both in the dark and after photoexcitation, Accessibility data
show that the rhodopsin polypeptide crosses an aqueous/hydrophobic bou
ndary near V138 and H152. The nitroxide mobilities inferred from the s
pectra are consistent with a model where the C helix extends to at lea
st residue C140, with much of the helix surface in contact with protei
n 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 invol
ves a rigid body movement of the C helix relative to the others in the
helix bundle.