Conformational changes at the carboxyl terminus of Gα occur during G protein activation

Abstract

To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Gα were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Gα(t)Gα(i1) determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (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 Cys210, AlF4/- caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4/- dependent increase in LY fluorescence (91%), indicating that Gα 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 Cys347 suggesting that Gα activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Gα and activated receptors, and may contribute to dissociation of activated Gα subunit from activated receptor.