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Nucleotides for G-Protein Signaling

Many signal transduction processes are regulated by GTP binding proteins (G-proteins) like small GTPases → Proteins of the Ras superfamily or heterotrimeric G-proteins → Proteins. The G-proteins act as molecular switches cycling between an inactive GDP-bound and an active GTP-bound state[1].

GTP and GDP analogs with various modifications have become indispensable tools to study G-protein signaling. In general, GTP and GDP analogs containing a modified phosphate moiety are resistant to enzymatic hydrolysis or are hydrolyzed at much smaller rates compared to their natural counterparts (non-hydrolyzable nucleotides).

2'/3'-Mant-,TNT- and Ant-modified GTP and GDP analogs combine fluorescence with close mimicry of the properties of natural nucleotides in respect of protein binding and interaction. They display environmentally sensitive fluorescence and therefore give strong signals upon a binding event which makes them perfect probes for stopped-flow and equilibrium analysis.

FAM-labeled GTP analogs have been shown to be an alternative for fluorescence anisotropy assays [2].

Table 1: GTP and GDP analogs for G-protein signaling analysis.

GTP AnalogModification
Mant-GTPribose moiety (2'/3'-OH)
fluorescent γ-[(4-Aminobutyl)triazolo]-GTP-5-FAM γ-phosphate moiety
Non-hydrolyzableGTPαSα-phosphate moiety
GTPγSγ-phosphate moiety
GpCppα, β-phosphate moiety
GppCpβ, γ-phosphate moiety

* These nucleotides possess both non-hydrolyzable and intrinsically fluorescent properties.

Selected References

[1] Vetter et al. (2001) The guanine nucleotide-binding switch in three dimensions. Science 294 (5545):1299.

[2] Pepanian et al. (2022) A fluorescence anisotropy assay with guanine nucleotides provides access to functional analysis of Gαi1 proteins. Anal Chem. 94(41):14410.