Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: Proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is p resumed to be the key to the restriction of binding of transducin and rhodo psin kinase to rhodopsin. Upon light-activation, this tertiary structure un dergoes a conformational change to form a new structure, which is recognize d by the above proteins and signal transduction is initiated. In this and t he following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach , C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-1248 5], we probe the dark state cytoplasmic domain structure in rhodopsin by in vestigating proximity between amino acids in different regions of the cytop lasmic face. The approach uses engineered pairs of cysteines at predetermin ed positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original ai-nino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic seque nce connecting helices I-II, disulfide bond formation was studied under str ictly defined conditions and plotted as a function of the position of the v ariable cysteines. An absolute maximum was observed for position 65 with tw o additional relative maxima for cysteines at positions 61 and 68. The obse rved disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small b ut significant motions are required for productive disulfide bond formation . During these motions, secondary structure elements are retained as indica ted by the lack of disulfide bond formation in cysteines that do not face t oward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.