Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin Cn is the first ev ent in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we u se a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicyl amide, The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio gro up, and the derivatized rhodopsin then is complexed with T by illumination at lambda > 495 nm. Subsequent irradiation of the complex at lambda 310 nm generates covalent crosslinks between the two proteins. Crosslinking was de monstrated between T and a number of single cysteine rhodopsin mutants, How ever, sites of crosslinks were investigated in detail only between T and th e rhodopsin mutant S240C (cytoplasmic loop V-VI), Crosslinking occurred pre dominantly with T-alpha. For identification of the sites of crosslinks in T -alpha the strategy used involved: (i) derivatization of all of the free cy steines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction o f the disulfide bond linking the two proteins and isolation of all of the T -alpha species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degra dation of the resulting T-alpha derivatives and isolation of T-alpha peptid es carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; an d (v) identification of the isolated peptides by matrix-assisted laser deso rption/ionization time-of-flight mass spectrometry. We found that crosslink ing occurred mainly to two C-terminal peptides in T-alpha containing the am ino acid sequences 310-313 and 342-345.