Dataset Information

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

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 ¹⁹F-labeling to explore the membrane-associated states of H-Ras in living cells. The site-specific incorporation of p-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 delivered ¹⁹F-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 ¹⁹F-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.

SUBMITTER: Ikari M

PROVIDER: S-EPMC10302746 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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

Ikari Masaomi M Yagi Hiromasa H Kasai Takuma T Inomata Kohsuke K Ito Masahiro M Higuchi Kae K Matsuda Natsuko N Ito Yutaka Y Kigawa Takanori T

JACS Au 20230601 6

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 19F-labeling to explore the membrane-associated states of H-Ras in living cells. The site-specific incorporation of p-trifluoromethoxyphenylalanine (OCF3P ...[more]

PMID: 37388687

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Project description:Cholesterol's effects on Na+,K+-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na+,K+-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na+,K+-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-β-cyclodextrin (mβCD). The mβCD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa3 + → E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K2 + → E1Na3 +). However, cholesterol depletion did not affect the observed rate constant for K+ occlusion by phosphorylated Na+,K+-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K2 +). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na+,K+-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na+,K+-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na+,K+-ATPase steady-state activity.

Dataset Information

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

Publications

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

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