Fluorescent guanine nucleotide analogs and G protein activation

Ann E. Remmers, Richard G Posner, Richard R. Neubig

Research output: Contribution to journalArticle

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Abstract

The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTPγS (mGTPγS) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleotide fluorescence was observed with excitation at 280 nm compared with 350 nm, due to energy transfer from tryptophan in G(o). G(o)-bound mGTPγS displays a blue-shift in its emission spectrum indicating a nonpolar environment for the G(o)-bound MANT. For the hydrolyzable analog, mGTP, the increase in fluorescence is followed by a decrease as it is hydrolyzed to mGDP. Unexpectedly, dissociation of mGDP was fast (t(1/2) 1.7 s) by comparison with GDP itself (t(1/2) 120 s). Binding of mGTPγS to G(o) was slow, but mastoparan increased the rate approximately 4-fold. For mGTP, mastoparan increased both the rate of binding and the peak fluorescence, even at saturating mGTP concentrations. Modeling the mGTP fluorescence kinetics in the presence and absence of mastoparan results in two novel conclusions. First, mGTP does not fully activate the G protein, even when bound. Second, mastoparan appears to increase the rate of the G protein conformational activation step, in addition to its known effect on GDP release.

Original languageEnglish (US)
Pages (from-to)13771-13778
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number19
StatePublished - May 13 1994

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Guanine Nucleotides
Guanosine Triphosphate
GTP-Binding Proteins
Chemical activation
Fluorescence
Energy Transfer
Fluorescent Dyes
Tryptophan
Energy transfer
Magnesium
Brain
Kinetics
mastoparan

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fluorescent guanine nucleotide analogs and G protein activation. / Remmers, Ann E.; Posner, Richard G; Neubig, Richard R.

In: Journal of Biological Chemistry, Vol. 269, No. 19, 13.05.1994, p. 13771-13778.

Research output: Contribution to journalArticle

Remmers, AE, Posner, RG & Neubig, RR 1994, 'Fluorescent guanine nucleotide analogs and G protein activation', Journal of Biological Chemistry, vol. 269, no. 19, pp. 13771-13778.
Remmers, Ann E. ; Posner, Richard G ; Neubig, Richard R. / Fluorescent guanine nucleotide analogs and G protein activation. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 19. pp. 13771-13778.
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