Elsevier

Methods

Volume 37, Issue 2, October 2005, Pages 183-189
Methods

Real-time in vitro measurement of GTP hydrolysis

https://doi.org/10.1016/j.ymeth.2005.05.019Get rights and content

Abstract

Small GTPases require an active GTPase activity to function correctly in their cellular environment. Mutation of key residues involved in this activity renders the GTPase defective and the small G-protein constitutively active (GTP-locked). The GTPase activity is also a target for GTPase-activating proteins (GAPs) which act to attenuate GTPase signalling by accelerating the conversion of bound GTP to bound GDP. The measurement of GTP hydrolysis in vitro can therefore provide information on the intrinsic activity of the small GTPase (e.g., mutated GTPase activity) as well as help define GAP specificity. Current methods to measure GTP hydrolysis in vitro utilise either radioactivity-based filter-binding assays or measurements of GDP:GTP:Pi ratios by high-performance liquid chromatography (HPLC). Both provide timed snapshots of the current GTP-bound state, can be prone to experimental errors, and do not provide a real-time observation of GTP hydrolysis. The method we describe here utilises a fluorescently labelled, phosphate-binding protein (PBP), which scavenges for free inorganic phosphate (Pi). On binding of a single Pi, a change of protein conformation is coupled to a 7-fold increase in fluorescence of the fluorophore. This method therefore permits real-time monitoring of GTPase activity, through measurement of Pi production. This review describes the process of preparing and labelling the PBP with the MDCC fluorophore, as well as an example of its use in measuring the GTPase activity of small GTPases. We also discuss the pros and cons, and implications of the technique in comparison to the radioactive and HPLC method of measuring the GTPase activity.

Introduction

Small GTPases are molecular switches which regulate diverse cellular functions that include the control of extracellular signal-mediated signal transduction (e.g., Ras and Rho family GTPases), vesicular transport (e.g., Rab, Arf, and Sar small GTPases), and nuclear transport (e.g., Ran small GTPase) signalling pathways [1]. Their intrinsic ability to bind GTP and GDP nucleotides, and to hydrolyse GTP to GDP, permits the cycling between the two different GTP (‘on’) and GDP (‘off’) nucleotide states. These two nucleotide-bound states provide the key different structural conformations which are recognised by small GTPase effector proteins, thus providing a mechanism for effector specificity to the GTP-bound form [2].

The intrinsic GTPase activity of small GTPases is critical in the attenuation of the small GTPase and its biochemical function, and acts as the antithesis to nucleotide exchange. The intrinsic GTPase rate of small GTPases is too slow to be of physiological significance in cellular signalling, thus GTPase-activating proteins (GAPs) act as molecular catalysts and accelerate GTP hydrolysis to a rate sufficient for maintaining a transient signal (often by up to 1 × 105)—members of the Ras and Rho family small GTPases are each regulated by multiple GAPs [3].

Although small GTPase GDP/GTP regulation is most commonly ascribed to extracellular stimulus activation of guanine nucleotide exchange factors, GAPs are clearly critical for maintaining proper GTPase function. For example, mutation of residues key in GTP hydrolysis (e.g., missense mutation of the conserved glutamine residue in Ras and G α heterotrimeric proteins), and, less so, the inactivation of GAPs (e.g., mutational loss of NF1, TSC or DLC-1), has been shown to cause constitutive small GTPase activation and persistent signalling, and contributing to cancer and other human diseases [3], [4], [5]. The ability to measure rates of GTP hydrolysis therefore can provide information on the intrinsic hydrolysis rate, the GAP-catalysed rate (and therefore GAP specificity), and mutation-impaired rate of a small GTPase’s GTP hydrolysis. It can also be used to gain an approximation of the binding constant between an effector and a small GTPase.

The phosphate probe described in this article was initially developed for analysing the actomyosin subfragment 1 ATPase [6] both in vitro and in situ. The phosphate-binding protein (PBP) is an endogenous Escherichia coli protein, and product of the phoS gene which is induced in times of Pi starvation [7]. Fortuitously for kinetic studies, PBP binds to Pi both rapidly (1.36 × 108 M−1 s−1) and tightly (Kd 0.1 μM), therefore ensuring that it is Pi release, rather than Pi binding that is being measured. Brune et al. introduced a cysteine to the lip of the Pi-binding cleft of PBP (A197C) allowing simple attachment of the coumarin fluorophore N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) [6], [8]. On binding of Pi in the cleft, a conformational change causes a 7-fold increase in MDCC fluorescence, which is measurable in a normal fluorimeter. The E. coli strain ANCC75, with pBR322-derived plasmid containing the A197C phosphate-binding protein, is available from Dr. Martin Webb (email: [email protected]) at the National Institute for Medical Research, Mill Hill, London, under an academic materials transfer agreement. MDCC is available from Molecular Probes (D10253).

The use of MDCC-PBP provides several advantages over the well-established radioactive filter-binding assay [9] and measurements by HPLC. Measurements can be made in real time with a regular fluorimeter, with no hazards or complicated steps (which may introduce significant experimental errors involved in the sample-taking for the filter-binding assay or HPLC). The method is also extremely simple and quick to perform.

PBP is extremely stable, and once conjugated to MDCC, can be stored at −80 °C for years. Indeed, the amounts produced from one successful PBP prep are sufficient for many years worth of experiments. The MDCC fluorophore is susceptible to photobleaching if subjected to intense light for too long. However, if the correct controls are performed (PBP alone) this can be accounted for during data analysis. In addition, modern fluorimeters are able to shut off the exciting light for periods, and so if a long time period is required, single point measurements can be taken and the light turned off so as to prevent photobleaching.

Section snippets

Preparation of phosphate-binding protein

The preparation and purification of PBP is a 3-day process. Labelling and further purification takes 1 day. In many ways it is similar to a standard protein preparation. However, in order to induce the bacteria to generate large amounts of PBP, bacteria are exposed to a ‘low’ phosphate growth medium. This growth condition is sufficient to cause significant PBP production. Once the PBP is produced, the outer bacterial cell wall is removed by an osmotic shock and the PBP purified by anionic

Data analysis

Once the data are recorded, it should be imported into a spreadsheet (Microsoft Excel) and then a data analysis application (ProFit for Mac OSX [http://www.quansoft.com/], Kaleidagraph for Windows or Mac OSX [http://www.synergy.com/]) for normalisation and curve fitting. It is possible to plot the raw data directly, however, if the data are normalised to the levels of 100% hydrolysed GTP (the maximum value of the data) comparison between different GTPases and conditions is easier. Data

Concluding remarks

The MDCC-PBP probe provides us with an easy method to examine GTP hydrolysis by GTPases in vitro, in real time. The measurement of the GTP hydrolysis rate not only provides us with information on the intrinsic rate, but can also be used to examine GAP specificities (which accelerate hydrolysis), as well as provide an estimation of binding constants between effectors (the binding of which inhibits hydrolysis). In this report, we have described the preparation of MDCC-PBP and its use in measuring

Acknowledgments

Our research was supported by grants from the National Institutes of Health (CA63071, 92240, and GM65533) and the Susan G. Komen Breast Cancer Foundation (to A.S.).

References (14)

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