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

[Image: see text] 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) spectr...

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Autores principales: Ikari, Masaomi, Yagi, Hiromasa, Kasai, Takuma, Inomata, Kohsuke, Ito, Masahiro, Higuchi, Kae, Matsuda, Natsuko, Ito, Yutaka, Kigawa, Takanori
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10302746/
https://www.ncbi.nlm.nih.gov/pubmed/37388687
http://dx.doi.org/10.1021/jacsau.3c00108
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author Ikari, Masaomi
Yagi, Hiromasa
Kasai, Takuma
Inomata, Kohsuke
Ito, Masahiro
Higuchi, Kae
Matsuda, Natsuko
Ito, Yutaka
Kigawa, Takanori
author_facet Ikari, Masaomi
Yagi, Hiromasa
Kasai, Takuma
Inomata, Kohsuke
Ito, Masahiro
Higuchi, Kae
Matsuda, Natsuko
Ito, Yutaka
Kigawa, Takanori
author_sort Ikari, Masaomi
collection PubMed
description [Image: see text] 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 α5, 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.
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spelling pubmed-103027462023-06-29 Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy Ikari, Masaomi Yagi, Hiromasa Kasai, Takuma Inomata, Kohsuke Ito, Masahiro Higuchi, Kae Matsuda, Natsuko Ito, Yutaka Kigawa, Takanori JACS Au [Image: see text] 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 α5, 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. American Chemical Society 2023-06-01 /pmc/articles/PMC10302746/ /pubmed/37388687 http://dx.doi.org/10.1021/jacsau.3c00108 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Ikari, Masaomi
Yagi, Hiromasa
Kasai, Takuma
Inomata, Kohsuke
Ito, Masahiro
Higuchi, Kae
Matsuda, Natsuko
Ito, Yutaka
Kigawa, Takanori
Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy
title Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy
title_full Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy
title_fullStr Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy
title_full_unstemmed Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy
title_short Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy
title_sort direct observation of membrane-associated h-ras in the native cellular environment by in-cell (19)f-nmr spectroscopy
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10302746/
https://www.ncbi.nlm.nih.gov/pubmed/37388687
http://dx.doi.org/10.1021/jacsau.3c00108
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