Characterization of heterotrimeric nucleotide-depleted Gαi-proteins by Bodipy-FL-GTPγS fluorescence anisotropy

Recombinant heterotrimeric G-protein αi1, αi2 and αi3 subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged β1γ2 subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted αi were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg2+, Mn2+ inhibited nucleotide binding to all αi-heterotrimers studied and accelerated nucleotide release. Mn2+ had stabilizing effect on the nucleotide free state of the αi1 subunit, whereas both Mn2+ as well as G-protein activation by mastoparan destabilized the αi2 subunit.