FORMATION OF THE META-II PHOTOINTERMEDIATE IS ACCOMPANIED BY CONFORMATIONAL-CHANGES IN THE CYTOPLASMIC SURFACE OF RHODOPSIN

Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in th e cytoplasmic domain. The single reactive cysteines were derivatized w ith a sulfhydryl-specific nitroxide spin-label, and the electron param agnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rat e of the attached nitroxides with polar and nonpolar paramagnetic agen ts indicated that they were all exposed to the aqueous environment. Ph otobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the at tached nitroxide. On the other hand, photobleaching in lauryl maltosid e produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These da ta directly reveal a light-induced conformational change in the cytopl asmic loops that accompanies meta II formation.