Masaomi Ikari, Hiromasa Yagi, Takuma Kasai, Kohsuke Inomata, Masahiro Ito, Kae Higuchi, Natsuko Matsuda, Yutaka Ito, Takanori Kigawa (2023) Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy., JACS Au, Volume 3, Number 6, pp. 1658-1669
Published in 2023 Jun 26 (Electronic publication in June 1, 2023, midnight )
(Abstract) 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-specific (19)F-labeling to 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., Tyr32 in switch I, Tyr96 interacting with switch II, and Tyr157 on helix alpha5, allowed the characterization of their conformational states depending on the nucleotide-bound states and an oncogenic mutational state. Exogenously delivered (19)F-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 three (19)F-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.