MAPPING LIGHT-DEPENDENT STRUCTURAL-CHANGES IN THE CYTOPLASMIC LOOP CONNECTING HELIX-C AND HELIX-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 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.