Conformational Changes at The Carboxyl Terminus of Gα Occur during G Protein Activation

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 Gαt/Gαi1 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 Cys347suggesting 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.