Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell ¹⁹F-NMR Spectroscopy

Ras acts as a molecular switch to control intracellularsignalingon the plasma membrane (PM). Elucidating how Ras associates with PMin the native cellular environment is crucial for understanding itscontrol mechanism. Here, we used in-cell nuclear magnetic resonance(NMR) spectroscopy combined with site-specific F-19-labelingto explore the membrane-associated states of H-Ras in living cells.The site-specific incorporation of p-trifluoromethoxyphenylalanine(OCF(3)Phe) at three different sites of H-Ras, i.e., Tyr32in switch I, Tyr96 interacting with switch II, and Tyr157 on helix alpha 5, allowed the characterization of their conformational statesdepending on the nucleotide-bound states and an oncogenic mutationalstate. Exogenously delivered F-19-labeled H-Ras proteincontaining a C-terminal hypervariable region was assimilated via endogenousmembrane-trafficking, enabling proper association with the cell membranecompartments. Despite poor sensitivity of the in-cell NMR spectraof membrane-associated H-Ras, the Bayesian spectral deconvolutionidentified distinct signal components on three F-19-labeledsites, thus offering the conformational multiplicity of H-Ras on thePM. Our study may be helpful in elucidating the atomic-scale pictureof membrane-associated proteins in living cells.