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

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Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell 19F-NMR Spectroscopy
JACS Au ( IF 8.5 ) Pub Date : 2023-06-01 , DOI: 10.1021/jacsau.3c00108
Masaomi Ikari ₁ , Hiromasa Yagi ₁ , Takuma Kasai _{1,

2} , Kohsuke Inomata _{1,

2} , Masahiro Ito ₁ , Kae Higuchi ₁ , Natsuko Matsuda _{1,

3} , Yutaka Ito ₄ , Takanori Kigawa ₁

Affiliation

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.

中文翻译：

通过细胞内 19F-NMR 光谱直接观察天然细胞环境中膜相关的 H-Ras

Ras 作为分子开关来控制质膜 (PM) 上的细胞内信号传导。阐明 Ras 如何在天然细胞环境中与 PM 关联对于理解其控制机制至关重要。在这里，我们使用细胞内核磁共振 (NMR) 光谱结合位点特异性¹⁹ F 标记来探索活细胞中 H-Ras 的膜相关状态。对三氟甲氧基苯丙氨酸 (OCF ₃ Phe) 在 H-Ras 的三个不同位点（即开关 I 中的 Tyr32、与开关 II 相互作用的 Tyr96 以及螺旋 α5 上的 Tyr157）进行位点特异性掺入，可以根据关于核苷酸结合状态和致癌突变状态。外源性传递¹⁹含有 C 末端高变区的 F 标记的 H-Ras 蛋白通过内源性膜运输被同化，从而能够与细胞膜区室正确关联。尽管膜相关 H-Ras 的细胞内 NMR 谱的灵敏度较差，但贝叶斯谱解卷积在三个¹⁹ F 标记位点上识别出不同的信号分量，从而提供了 PM 上 H-Ras 的构象多样性。我们的研究可能有助于阐明活细胞中膜相关蛋白的原子尺度图像。

更新日期：2023-06-01

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