A direct fluorescence-based assay for RGS domain GTPase accelerating activity
Section snippets
Materials
BODIPYFL-GTP and guanosine 5′-O-(3-thiotriphosphate), BODIPYFL thioester (BODIPYFL-GTPγS) were obtained from Molecular Probes (Eugene, OR, USA). Unless otherwise specified, all other chemicals were of the highest purity obtainable from Sigma (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).
Cloning
A prokaryotic expression construct encoding an N-terminal GST fusion of the extended RGS domain of rat RGS14 (aa 57–235) was created using the pGEX4TEV2 vector modified from pGEX4T2 (Amersham
Results
We examined the suitability of the ribose-conjugated GTP analog BODIPYFL-GTP (Fig. 1A) as a fluorescent spectroscopic probe for GTP binding and hydrolysis by heterotrimeric G protein α subunits. As shown previously [18], BODIPYFL-GTP undergoes time-dependent changes in fluorescence on interaction with Gαo (Fig. 2A). This response is biphasic, with an initial increase in fluorescence corresponding to GTP binding followed by decay in fluorescence corresponding to GTP hydrolysis by Gαo (for a
Discussion
We have described a direct real-time assay for Gαo GTPase activity with specific applications to the measurement of RGS protein GAP activity. BODIPYFL-GTP undergoes a 200% increase in fluorescence quantum yield when bound to Gαo, and hydrolysis of this nucleotide to its GDP form decreases its fluorescence to 27% above its unbound value. Thus, BODIPYFL-GTP represents a useful fluorescence biosensor for GTPase activity and therefore G-protein activation state.
Among the series of described
Acknowledgments
The authors thank Melinda Hains and Christopher McCudden for critical appraisal of the manuscript. We also thank Randall J. Kimple and Breann L. Wolfe (UNC Pharmacology) for assistance with RGS14 vector construction and GST-RGS14 purification, respectively. Francis Willard is an American Heart Association postdoctoral fellow. This research was supported by National Institutes of Health grants (R01 GM062338 and P01 GM065533).
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