19F and 31P NMR spectroscopy of G protein alpha subunits. Mechanism of activation by Al3+ and F-.
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.
Higashijima
T
T
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Graziano
M P
MP
Suga
H
H
Kainosho
M
M
Gilman
A G
AG
D000535:Aluminum / Q000737:chemistry*; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D005459:Fluorides / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010759:Phosphorus Isotopes; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D010759:Phosphorus Isotopes; D014364:Tryptophan; D000535:Aluminum; D019204:GTP-Binding Proteins; D008274:Magnesium; D005459:Fluorides