NMR Detection Methods for Profiling RAS Nucleotide Cycling.

Abstract

RAS oncoproteins exhibit a switch-like behavior to drive diverse signaling cascades. In the active GTP-bound state, a conformational change occurs in these enzymes that enables interaction with downstream effectors. Nucleotide-dependent conformational exchange is easily detected with real-time NMR (RT-NMR) spectroscopy. RT-NMR has been firmly established as an effective assay to measure RAS oncoprotein nucleotide exchange and GTP hydrolysis kinetics and can further determine the regulatory activity of guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). It is now possible to multiplex these assays, allowing for the precise monitoring of activation states for mixtures of RAS oncoproteins or other RAS superfamily GTPases. Here, we describe the protocols necessary to express and purify isotopically labeled RAS and detail how to carry out an RT-NMR assay on a singular RAS protein or on a mixture of small GTPases.