To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on G alpha were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant G alpha(t)/G alpha(il) determined that two cysteines are the major fluorescent labeling sites, Cys(210), located in the switch II region, and Cys(347) at the C terminus. Mutants with serines replacing Cys(210) (Chi6a) and Cy-347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys(210), AlF4- caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys(210) is a reporter of conformational change in the switch II region. Chi6a labeled at Cys(347) also showed an AlF4--dependent increase in LY fluorescence (91%), indicating that G alpha activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4- before labeling led to a decreased incorporation of LY into Cys(347) suggesting that G alpha activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on G alpha and activated receptors, and may contribute to dissociation of activated G alpha subunit from activated receptor.
|Autori:||Yang C; Skiba N; Mazzoni M; Hamm H|
|Titolo:||Conformational changes at the carboxyl terminus of G alpha occur during G protein activation|
|Anno del prodotto:||1999|
|Digital Object Identifier (DOI):||10.1074/jbc.274.4.2379|
|Appare nelle tipologie:||1.1 Articolo in rivista|