Structure and function in rhodopsin: Effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase

Six rhodopsin mutants containing disulfide cross-links between different cy toplasmic regions were prepared: disulfide bond 1, between Cys65 (interheli cal loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys 246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, betw een Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfid e bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); a nd disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminu s), The effects of local restrictions caused by the cross-links on transduc in (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond I showed Little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative mot ion between interhelical loop I-II and helix VII is not crucial for recogni tion by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK, Disulfide bond 6 resulted in enhance d G(T) activation but abolished phosphorylation by RK, suggesting the struc ture recognized by G(T) was stabilized in this mutant by cross-linking of t he C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction, In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic f ace that are necessary for protein-protein interactions need not be coopera tive, and may be segmental.