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.
University of Tartu, Institute of Chemistry, Ravila 14a, 50411 Tartu, Estonia.
Lauri
L
Tõntson
Anna
A
Babina
Taavi
T
Võsumaa
Sergei
S
Kopanchuk
Ago
A
Rinken
D000818:Animals; D002460:Cell Line; D005454:Fluorescence Polarization; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D014862:Wasp Venoms / Q000494:pharmacology
C486958:BODIPY FL GTPgammaS; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D021122:Protein Subunits; D014862:Wasp Venoms; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; C037500:mastoparan; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D008274:Magnesium