Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases

doi:10.1016/j.ab.2008.06.039

Analytical Biochemistry

Volume 381, Issue 2, 15 October 2008, Pages 258-266

https://doi.org/10.1016/j.ab.2008.06.039 Get rights and content

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 Mg²⁺, 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 γ-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.

Section snippets

Reagents

All reagents were of analytical quality and, unless otherwise noted, were obtained from Sigma (St. Louis, MO, USA). Plasticware was obtained from VWR (West Chester, PA, USA). Sephadex G-25, glutathione (GSH) Sepharose 4B, and Superdex peptide beads were purchased from Amersham Biosciences (Piscataway, NJ, USA); the Superdex peptide beads were 13 μm with an exclusion limit of 7000 Da and were extruded from a column. Purification and filter concentrators were purchased from Millipore (Billerica,

Purified GTPases can be immobilized on GSH beads, and GTP binding can be detected by flow cytometry

We previously demonstrated the utility of flow cytometry for accurately measuring the dynamics of interaction between GST–GFP fusion proteins and GSH beads [30]. Here we extended the utility of GSH beads to the characterization of the equilibrium binding and association and dissociation kinetics between GST–Rab7 and BODIPY FL-tagged guanine nucleotides. The molecular assembly schemes are shown in Eqs. (2), (3). The first step entails binding of purified GST–Rab7 (Fig. 1A) to the GSH beads

Discussion

We report a new bead-based flow cytometry assay to measure in real time the affinity and kinetics of nucleotide binding to small GTPases using GST fusions of Rab7 and RhoA as test cases. The particular features of the assay that ensure broad utility are the facts that (i) the assay circumvents the need for radiolabeled analogues, (ii) only femtomoles of purified protein are needed per assay, (iii) the assay may be run on a conventional flow cytometer, (iv) the assay is quick and measures

Acknowledgments

This work is generously supported by the National Science Foundation (MCB0446179) to A.W-N. and National Institutes of Health (R03MH081231 to A.W-N., K25AI60036 to T.B., and CA1181000 and MH074425 to L.A.S.). M.T. was supported by a University of New Mexico (UNM) Research and Allocations (RAC) postdoctoral fellow award. O.P. and A.R. were supported by a European Young Investigator Award to A.R. We gratefully acknowledge Elsa Romero, Jacob Agola, and Mark Carter for technical assistance as well

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¹: Authors contributed equally to the work.

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Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases

Abstract

Section snippets

Reagents

Purified GTPases can be immobilized on GSH beads, and GTP binding can be detected by flow cytometry

Discussion

Acknowledgments

Trends Cell Biol.

Adv. Drug Deliv. Rev.

J. Biol. Chem.

Curr. Biol.

J. Biol. Chem.

Anal. Biochem.

Methods

Methods Enzymol.

J. Struct. Biol.

Trends Cell Biol.

Methods Enzymol.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Chromatogr. B

Anal. Biochem.

Anal. Biochem.

Small GTP-binding proteins

Physiol. Rev.

The Ras superfamily at a glance

J. Cell Sci.

Rab GTPases at a glance

J. Cell Sci.

Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome

Nat. Genet.

Charcot–Marie–Tooth disease: An update

Curr. Opin. Neurol.