Ras acts as a molecular switch to control intracellular
signaling
on the plasma membrane (PM). Elucidating how Ras associates with PM
in the native cellular environment is crucial for understanding its
control mechanism. Here, we used in-cell nuclear magnetic resonance
(NMR) spectroscopy combined with site-specific19F-labeling
to explore the membrane-associated states of H-Ras in living cells.
The site-specific incorporation ofp-trifluoromethoxyphenylalanine
(OCF3Phe) at three different sites of H-Ras, i.e., Tyr32
in switch I, Tyr96 interacting with switch II, and Tyr157 on helix
α5, allowed the characterization of their conformational states
depending on the nucleotide-bound states and an oncogenic mutational
state. Exogenously delivered19F-labeled H-Ras protein
containing a C-terminal hypervariable region was assimilated via endogenous
membrane-trafficking, enabling proper association with the cell membrane
compartments. Despite poor sensitivity of the in-cell NMR spectra
of membrane-associated H-Ras, the Bayesian spectral deconvolution
identified distinct signal components on three19F-labeled
sites, thus offering the conformational multiplicity of H-Ras on the
PM. Our study may be helpful in elucidating the atomic-scale picture
of membrane-associated proteins in living cells.