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

Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at the palmitoylation sites at 322C and 323C. The substituted cysteine residues were modified with a selective reagent to generate a nitroxide side chain, and the electron paramagnetic resonance spectrum of each spin-labeled mutant was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 306-314 were in a regular alpha-helical conformation representing the end of helix VII. This helix apparently extends about 1.5 turns above the surface of the membrane, with one face in strong tertiary interaction with the core of the protein. For the segment 315-321, substituted cysteine residues at 317, 318, 320, and 321 had low reactivity with the spin-label reagent. This segment has the most extensive tertiary interactions yet observed in the rhodopsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine residues at 65 and 316. This result indicates that at least some of the tertiary contacts made in the 315-321 segment are with the sequence connecting transmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 313 can be accounted for by movements in the adjacent helix VI.

The cytoplasmic interhelical E-F loop in rhodopsin is a part of the region that interacts with the G-protein transducin and rhodopsin kinase during signal transduction. In extending the previous work on systematic single cysteine substitutions of the amino acids in the cytoplasmic C-D loop, we have now replaced, one at a time, the amino acids Q225-I256 in the E-F loop region by cysteines. All the mutants formed the characteristic rhodopsin chromophore with 11-cis-retinal. While most of the mutants bleached normally, L226C, showed abnormal bleaching behavior. A study of the alkylation of the mutants by N-ethylmaleimide in dark showed low reactivity by some mutants, especially L226C. The rates of transducin activation (GT(alpha)-GTP gamma S complex formation) were measured for all the mutants. While these were normal for the bulk of the mutants, some (L226C, T229C, V230C, A233C, A234C, T242C, T243C, and Q244C) showed strikingly reduced transducin activation. The results suggest a specific structure in the E-F loop that interacts with transducin.

Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of arc on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI (Altenbach, C., Yang, K., Farrens, D., Farahbakhsh, Z., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470).