Real-time in vitro measurement of GTP hydrolysis.
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:P(i) 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 (P(i)). On binding of a single P(i), 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 P(i) 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.
Shutes
Adam
A
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC 27599-7295, USA. ashutes@med.unc.edu
Der
Channing J
CJ
D000818:Animals; D020558:GTP Phosphohydrolases / Q000032:analysis* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D028044:Phosphate-Binding Proteins; D013194:Staining and Labeling / Q000379:methods
D028044:Phosphate-Binding Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases