Paul Maxum E, Vish Kimberly J, Boggon Titus J
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Department of Pharmacology, Yale University, New Haven, CT 06510, USA; Yale Cancer Center, Yale University, New Haven, CT 06510, USA.
Methods. 2025 Sep 8;244:108-117. doi: 10.1016/j.ymeth.2025.09.003.
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.
Ras小GTP酶对广泛的细胞过程至关重要。这些蛋白质在结合GDP和结合GTP的状态之间循环，并且GTP酶激活蛋白（GAPs）的作用对于刺激Ras介导的GTP水解是必需的。在这里，我们提供了一种通过体外实时监测无机磷酸盐释放来实现对GAP介导的小GTP酶刺激进行米氏动力学分析的方案。这是通过磷酸盐传感器蛋白的荧光来实现的，该蛋白是一种与周质磷酸盐结合蛋白（PstS）结合的MDCC共轭物。我们以预先加载GTP的H-Ras小GTP酶及其受p120RasGAP（RasGAP，RASA1）刺激为例来说明该方案。我们讨论了方案设计、测定方法开发、数据收集、处理和分析。典型的测定包括多达二十个同时进行的反应，磷酸盐生成速率约为数十nM/s。我们还提供了优化试剂条件（特别是盐浓度）的指南，并评估了它们的功能影响。所描述的方案提供了一种方便且全面的方法，使用广泛可用的材料来准确监测GAP蛋白对小GTP酶的激活，适用于一系列应用。
