ArticleAnalytical biochemistry2008
Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases.
Article in Analytical biochemistry, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 25 citations in OpenAlex.
- Phosphovariants of the canonical heterotrimeric Gα protein, GPA1, differentially affect G protein activity and Arabidopsis development.bioRxiv : the preprint server for biology · 2026Article
- GLOBAL AND TARGETED PROFILING OF GTP-BINDING PROTEINS IN BIOLOGICAL SAMPLES BY MASS SPECTROMETRY.Mass spectrometry reviews · 2021Review
- Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases.Methods in molecular biology (Clifton, N.J.) · 2018Article
- Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.Journal of biochemistry · 2016Article
- R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis.Molecular cancer therapeutics · 2015Article
- Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal protein (RILP) effector protein.Methods in molecular biology (Clifton, N.J.) · 2015Article
- A Pan-GTPase Inhibitor as a Molecular Probe.PloS one · 2015Article
- Article
- Rapid parallel flow cytometry assays of active GTPases using effector beads.Analytical biochemistry · 2013Article
- Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.The Journal of biological chemistry · 2013Article
- A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.ACS chemical biology · 2012Article
- High-throughput flow cytometry bead-based multiplex assay for identification of Rho GTPase inhibitors.Methods in molecular biology (Clifton, N.J.) · 2012Article
- A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1.Molecular biology of the cell · 2011Article
- Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay.Journal of biomolecular screening · 2010Article
- Large nucleotide-dependent conformational change in Rab28.FEBS letters · 2008Article
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Authors and funding
9 authors at 5 institutions in 3 countries.
Funding
Abstract
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.