19F and 31P NMR spectroscopy of G protein alpha subunits. Mechanism of activation by
 

19F and 31P NMR spectroscopy of G protein alpha subunits. Mechanism of activation by Al3+ and F-.

Related Articles 19F and 31P NMR spectroscopy of G protein alpha subunits. Mechanism of activation by Al3+ and F-.

J Biol Chem. 1991 Feb 25;266(6):3396-401

Authors: Higashijima T, Graziano MP, Suga H, Kainosho M, Gilman AG

19F and 31P NMR spectroscopy was used to study the mechanism of activation of the alpha 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 alpha subunits (G alpha) 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 alpha or with GDP alone. Titration of Al3+, Mg2+, and F- indicated that each molecule of G alpha binds 3-5 molecules of F- (Kd = 0.47 mM), a single molecule of Al3+ (Kd much less than 0.1 mM), and a single Mg2+ ion (Kd 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- (less than 1 nm). 31P NMR of GDP-G alpha showed peaks at -2 and -8.6 ppm, corresponding to the beta- and alpha-phosphoryl groups of GDP, respectively. Binding of Al3+, Mg2+, and F- caused an upfield shift of 6 ppm for the beta-phosphoryl signal with no change in the alpha-phosphoryl signal. These observations indicate that Mg2+.GDP.AlF3-5 mimics Mg2+.GTP in its capacity to activate G protein alpha subunits.

PMID: 1899863 [PubMed - indexed for MEDLINE]



Source: PubMed