Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan National Laboratory for Optoelectronics, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, 430071, China.
Angew Chem Int Ed Engl. 2023 May 22;62(22):e202300318. doi: 10.1002/anie.202300318. Epub 2023 Apr 20.
In-cell NMR spectroscopy is an effective tool for observing proteins at atomic resolution in their native cellular environment. However, its utility is limited by its low sensitivity and the extensive line broadening caused by nonspecific interactions in the cells, which is even more pronounced in human cells due to the difficulty of overexpressing or delivering high concentrations of isotopically labeled proteins. Here, we present a high-sensitivity tag (wPSP-6F) containing two trifluoromethyl groups that can efficiently label globular proteins with molecular weights in the 6-40 kDa range under mild conditions. This tag allowed us to detect globular proteins in human cells at concentrations as low as 1.0 μM, which would not have been achievable with N or 3-fluorotyrosine labeling. Moreover, we detected conformational changes and interactions of proteins in the cellular environment. The new sensitive F NMR tag may significantly expand the scope of protein NMR in human cells.
细胞内 NMR 光谱学是一种有效的工具，可在其天然细胞环境中以原子分辨率观察蛋白质。然而，其应用受到灵敏度低和细胞中非特异性相互作用引起的广泛线宽的限制，由于在人类细胞中过表达或递送电离标记的蛋白质的高浓度更加困难，这种限制更为明显。在这里，我们提出了一种含有两个三氟甲基基团的高灵敏度标签 (wPSP-6F)，可在温和条件下有效地标记分子量在 6-40 kDa 范围内的球状蛋白质。该标签允许我们以低至 1.0 μM 的浓度检测人类细胞中的球状蛋白质，这是使用 N 或 3-氟酪氨酸标记无法实现的。此外，我们还检测到了蛋白质在细胞环境中的构象变化和相互作用。新的灵敏 F NMR 标签可能会显著扩展蛋白质 NMR 在人类细胞中的应用范围。
