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

Contact sites in interaction between light-activated rhodopsin and transduc in (T) have been investigated by using a chemically preactivated crosslinki ng reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, The 3 propionyl-N -succinimidyl group in the reagent was attached by a disulfide exchange rea ction to rhodopsin mutants containing single reactive cysteine groups in th e cytoplasmic loops. Complex formation between the derivatized rhodopsin mu tants and T was carried out by illumination at lambda > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crossli nking of rhodopsin to the T-alpha subunit. Crosslinking to T-alpha was demo nstrated for the rhodopsin mutants K141C, S240C, and K248C. and the crossli nked sites in T-alpha were identified for the rhodopsin mutant S240C, The p eptides carrying the crosslinking moiety were isolated from the trypsin-dig ested peptide mixture, and their identification was carried out by matrix-a ssisted laser desorption ionization-time of flight mass spectrometry, The m ain site of crosslinking is within the peptide sequence, Leu-19-Arg-28 at t he N-terminal region of T-alpha. The total results show that both the N and the C termini of T-alpha are in close vicinity to the third cytoplasmic lo op of rhodopsin in the complex between rhodopsin and T.