¹⁹F and ³¹P NMR spectroscopy of G protein α subunits: Mechanism of activation by Al³⁺ and F^-

¹⁹F and ³¹P NMR spectroscopy was used to study the mechanism of activation of the α subunits of guanine nucleotide-binding regulatory proteins (G proteins) by Al³⁺, Mg²⁺, and F^-. ¹⁹F NMR spectra of solutions containing Al³⁺, Mg²⁺, and F^- showed a characteristic F^- peak at -10 ppm. Addition of the GDP-bound form of either of two G protein α subunits (G_α) resulted in the appearance of an additional peak at -29 or -30 ppm. This peak was not observed with guanosine 5′-3-O-(thio)triphosphate-G_α or with GDP alone. Titration of Al³⁺, Mg²⁺, and F^- indicated that each molecule of G_α binds 3-5 molecules of F^- (K_d = 0.47 mM), a single molecule of Al³⁺ (K_d ≪ 0.1 mM), and a single Mg²⁺ ion (K_d about 0.1 mM). Replacement of Mg²⁺ with Mn²⁺ caused a dramatic broadening of the NMR signal, indicating that the metal ion binds in proximity to the protein-bound F^- (<1 nm). ³¹P NMR of GDP-G_α showed peaks at -2 and -8.6 ppm, corresponding to the β- and α-phosphoryl groups of GDP, respectively. Binding of Al³⁺, Mg²⁺, and F^- caused an upfield shift of 6 ppm for the β-phosphoryl signal with no change in the α-phosphoryl signal. These observations indicate that Mg²⁺·GDP·AlF_3-5 mimics Mg²⁺·GTP in its capacity to activate G protein α subunits.