Several processes involving heterotrimeric G
E64d have been shown to be dependent on Mg2+ concentration. Very low Mg2+ concentrations (nM range) are required for GTPase activity, slow dissociation of GTPγS from α subunits, and intrinsic tryptophan fluorescence enhancement, while βγ-stimulated GDP dissociation and GTPγS induced subunit dissociation call for high Mg2+ concentrations (mM range) [23]. Two scenarios that have been proposed to explain the observed differences in Mg2+ concentrations required for G protein functions are: there is one Mg2+ site that has a fluctuating metal ion affinity which is dependent on the protein conformation, or there are two distinct Mg2+
binding sites with dissimilar affinities [23]. The data shown in Fig. 3 and summarized in Table 1 confirms that the latter occurs. Fig. 3 shows that the high affinity Mg2+ binding site is primarily populated as the α subunit is first titrated with Mg2+ while population of the low affinity site requires higher concentrations of Mg2+ relative to the α subunit concentration.
