Recent developments of in-cell NMR methodology enabled the observation of various intracellular events, including post-translational modifications, protein folding stabilities, and protein maturation at an atomic resolution. Key technologies enabling the in-cell NMR observation within the mammalian cells include the method for efficient protein incorporation and the bioreactor system for keeping cells under physiological condition during long-term NMR measurements.
We utilized the in-cell NMR method for real-time monitoring of the activation status of small GTPase RAS protein and its oncogenic mutants[1]. Time-resolved in-cell NMR experiments allow us to measure the time-dependent changes of the fraction of active, GTP-bound RAS under the cellular condition, thereby demonstrating that the intracellular GTP-bound form is lower than in vitro, due to the increase of GTP hydrolysis rate as well as the decrease of GDP-GTP exchange rates. We also established the in-cell NMR method to estimate the contribution of the specific regulator molecule of RAS using the knockout cells. We also used in-cell NMR to observe the progression of the reaction of covalent inhibitors of the KRAS G12C mutant in real time, thereby elucidating the mechanism of action of the inhibitors. These collective studies demonstrated the power of in-cell NMR for the biological and pharmacological research.