Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: A site-directed spin-labeling study

Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at th e 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 mutan t was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 30 6-314 were in a regular ct-helical conformation representing the end of hel ix 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 se gment has the most extensive tertiary interactions yet observed in the rhod opsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine resi dues at 65 and 316. This result indicates that at least some of the tertiar y contacts made in the 315-321 segment are with the sequence connecting tra nsmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 3 13 can be accounted for by movements in the adjacent helix VI.