Michaelis-Menten kinetics of RasGAP proteins by a rapid fluorescence-based assay

Ras small GTPases are essential for a wide range of cellular processes. These proteins cycle between the GDP-loaded and GTP-loaded states, and the actions of GTPase activating proteins (GAPs) are necessary to stimulate Ras-mediated GTP hydrolysis. Here, we provide a protocol to achieve Michaelis-Menten kinetic profiling of GAP-mediated stimulation of a small GTPase by real-time monitoring of inorganic phosphate release in vitro. This is achieved using fluorescence of the Phosphate Sensor protein, an MDCC conjugate with periplasmic phosphate binding protein (PstS). We use H-Ras small GTPase pre-loaded with GTP and its stimulation by p120RasGAP (RasGAP, RASA1) as an example of this protocol. We discuss protocol design, assay development, data collection, processing, and analysis. Typical assays comprise up to twenty simultaneous reactions with phosphate production rates on the order of tens of nM/s. We also provide guidelines for the optimization of reagent conditions, particularly salt concentrations, and assess their functional impact. The described protocol provides a convenient and comprehensive method to achieve accurate monitoring of small GTPase activation by GAP proteins using widely available materials and suitable to a range of applications.