Dynamics of Arrestin-Rhodopsin Interactions

Martha E. Sommer, David L. Farrens, J. Hugh McDowell, Lauren A. Weber, W. Clay Smith
2007 Journal of Biological Chemistry  
In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly
more » ... ng the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state. . 4 The abbreviations used are: Rho-P, phosphorylated rhodopsin; Rho*-P, lightactivated rhodopsin; max , wavelength of maximum absorption or emission; ex , wavelength of excitation; em , wavelength of emission; I72B, bimane-labeled arrestin I72C; DTNB, 5,5Ј-dithiobis(2,2Ј-nitrobenzoic acid); TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; DTT, dithiothreitol.
doi:10.1074/jbc.m702155200 pmid:17606620 fatcat:brhgr7ffovfprpbtmsvzguffb4