The G226A mutant of G(sα) highlights the requirements for dissociation of G protein subunits

Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form of G(sα) instead of the wild-type protein. The mutant G(sα) protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein α subunit from the βγ subunit complex (Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B., and Bourne, H.R. (1988) Nature 334, 712-715). G226A G(sα) was synthesized in Escherichia coli, purified, and characterized. Examination of the kinetics of dissociation of guanosine 5'-3-O-(thio)triphosphate (GTPγS) suggests that G226A G(sα) is incapable of assuming the conformation necessary for high affinity binding of Mg²⁺ to the α subunit-GTPγS complex. Associated changes include the failure of Mg²⁺ and GTPγS to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of α from βγ. However, the GTPase activity of the mutant protein is near normal (at high Mg²⁺ concentrations), and the protein is capable of activating adenylylcyclase. A similar defect is present in G49V G(sα). Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A G(sα), and this mutation thus highlights the crucial nature of this reaction as a component of G protein action.