19F and 31P NMR spectroscopy of G protein α subunits

Mechanism of activation by Al3+ and F-

Tsutomu Higashijima, Michael P. Graziano, Hinako Suga, Masatsune Kainosho, Alfred G. Gilman

Research output: Contribution to journalArticle

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Abstract

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 (Gα) 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-Gα or with GDP alone. Titration of Al3+, Mg2+, and F- indicated that each molecule of Gα binds 3-5 molecules of F- (Kd = 0.47 mM), a single molecule of Al3+ (Kd ≪ 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- (<1 nm). 31P NMR of GDP-Gα 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·AlF3-5 mimics Mg2+·GTP in its capacity to activate G protein α subunits.

Original languageEnglish (US)
Pages (from-to)3396-3401
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number6
StatePublished - Feb 25 1991

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Guanine Nucleotides
Protein Subunits
GTP-Binding Proteins
Nuclear magnetic resonance spectroscopy
Carrier Proteins
Magnetic Resonance Spectroscopy
Chemical activation
Nuclear magnetic resonance
Molecules
Ions
Guanosine 5'-O-(3-Thiotriphosphate)
Guanosine Triphosphate
Titration
Metal ions
Proteins
Metals

ASJC Scopus subject areas

  • Biochemistry

Cite this

Higashijima, T., Graziano, M. P., Suga, H., Kainosho, M., & Gilman, A. G. (1991). 19F and 31P NMR spectroscopy of G protein α subunits: Mechanism of activation by Al3+ and F-. Journal of Biological Chemistry, 266(6), 3396-3401.

19F and 31P NMR spectroscopy of G protein α subunits : Mechanism of activation by Al3+ and F-. / Higashijima, Tsutomu; Graziano, Michael P.; Suga, Hinako; Kainosho, Masatsune; Gilman, Alfred G.

In: Journal of Biological Chemistry, Vol. 266, No. 6, 25.02.1991, p. 3396-3401.

Research output: Contribution to journalArticle

Higashijima, T, Graziano, MP, Suga, H, Kainosho, M & Gilman, AG 1991, '19F and 31P NMR spectroscopy of G protein α subunits: Mechanism of activation by Al3+ and F-', Journal of Biological Chemistry, vol. 266, no. 6, pp. 3396-3401.
Higashijima T, Graziano MP, Suga H, Kainosho M, Gilman AG. 19F and 31P NMR spectroscopy of G protein α subunits: Mechanism of activation by Al3+ and F-. Journal of Biological Chemistry. 1991 Feb 25;266(6):3396-3401.
Higashijima, Tsutomu ; Graziano, Michael P. ; Suga, Hinako ; Kainosho, Masatsune ; Gilman, Alfred G. / 19F and 31P NMR spectroscopy of G protein α subunits : Mechanism of activation by Al3+ and F-. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 6. pp. 3396-3401.
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N2 - 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 (Gα) 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-Gα or with GDP alone. Titration of Al3+, Mg2+, and F- indicated that each molecule of Gα binds 3-5 molecules of F- (Kd = 0.47 mM), a single molecule of Al3+ (Kd ≪ 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- (<1 nm). 31P NMR of GDP-Gα 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·AlF3-5 mimics Mg2+·GTP in its capacity to activate G protein α subunits.

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