Real-Time Monitoring of RAS Activity Using In Vitro and In-Cell NMR Spectroscopy

Abstract

The activation level of RAS can be determined by GTP hydrolysis rate (k_hy) and GDP-GTP exchange rates (k_ex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the k_hy and k_ex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the k_ex and k_hy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the k_hy and k_ex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.

Keywords: GDP-GTP exchange rate; GTP hydrolysis rate; GTP regeneration system; In-cell NMR; RAS; Real-time NMR; Small GTPase.