Maxum E. Paul , Kimberly J. Vish , Titus J. Boggon
{"title":"Michaelis-Menten kinetics of RasGAP proteins by a rapid fluorescence-based assay","authors":"Maxum E. Paul , Kimberly J. Vish , Titus J. Boggon","doi":"10.1016/j.ymeth.2025.09.003","DOIUrl":null,"url":null,"abstract":"<div><div>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 <em>in vitro</em>. 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.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 108-117"},"PeriodicalIF":4.3000,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S104620232500194X","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
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.
期刊介绍:
Methods focuses on rapidly developing techniques in the experimental biological and medical sciences.
Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.