TOPOGRAPHIC STUDY OF ARRESTIN USING DIFFERENTIAL CHEMICAL MODIFICATIONS AND HYDROGEN-DEUTERIUM EXCHANGE

Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P-Rho). To study its conformational changes and regions interacting with P-Rho, arrestin w as subjected to (1) differential acetylation at lysine residues in the presence and absence of P-Rho, and (2) amide hydrogen/deuterium exch ange. Labeled protein was proteolysed and analyzed by mass spectrometr y. Three Lys residues, 28, 176, and 211, were protected from acetylati on in native arrestin, although they were not located in regions exhib iting slow amide hydrogen exchange rates. The presence of P-Rho prote cted lysine 201 from acetylation and partially protected 14 other lysy l residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), ( 267, 276), (298, 300), and 367, where parentheses indicate lysine resi dues found within the same peptide. In contrast, in the C-terminal reg ion of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P-Rho, These data allowed us to identify functional regio ns in the arrestin molecule.