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

Archives of Biochemistry and Biophysics, ISSN: 0003-9861, Vol: 524, Issue: 2, Page: 93-98
2012
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Article Description

Recombinant heterotrimeric G-protein α i1, α i2 and α i3 subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged β 1 γ 2 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 Mg 2+, Mn 2+ inhibited nucleotide binding to all α i -heterotrimers studied and accelerated nucleotide release. Mn 2+ had stabilizing effect on the nucleotide free state of the α i1 subunit, whereas both Mn 2+ as well as G-protein activation by mastoparan destabilized the α i2 subunit.

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