Characterization of heterotrimeric nucleotide-depleted Gα(i)-proteins by Bodipy-FL-GTPγS fluorescence anisotropy.

Recombinant heterotrimeric G-protein α(i1), α(i2) and α(i3) 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 α(i) 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 α(i)-heterotrimers studied and accelerated nucleotide release. Mn²⁺ had stabilizing effect on the nucleotide free state of the α(i1) subunit, whereas both Mn²⁺ as well as G-protein activation by mastoparan destabilized the α(i2) subunit.