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.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1991|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology