Movellan Kumar Tekwani, Zhu Wenkai, Banks Daniel, Kempf James, Runge Brent, Gronenborn Angela M, Polenova Tatyana
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, 1051 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA.
Sci Adv. 2024 Oct 4;10(40):eadq3115. doi: 10.1126/sciadv.adq3115. Epub 2024 Oct 2.
Obtaining atomic-level information on components in the cell is a major focus in structural biology. Elucidating specific structural and dynamic features of proteins and their interactions in the cellular context is crucial for understanding cellular processes. We introduce F dynamic nuclear polarization (DNP) combined with fast magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy as a powerful technique to study proteins in mammalian cells. We demonstrate our approach on the severe acute respiratory syndrome coronavirus 2 5F-Trp-N protein, electroporated into human cells. DNP signal enhancements of 30- to 40-fold were observed, translating into over 1000-fold experimental time savings. High signal-to-noise ratio spectra were acquired on nanomole quantities of a protein in cells in minutes. 2D F-F dipolar correlation spectra with remarkable sensitivity and resolution were obtained, exhibiting F-F cross peaks associated with fluorine atoms as far as ~10 angstroms apart. This work paves the way for F DNP-enhanced MAS NMR applications in cells for probing protein structure, dynamics, and ligand interactions.
获取细胞内成分的原子水平信息是结构生物学的一个主要研究重点。阐明蛋白质在细胞环境中的特定结构和动态特征以及它们之间的相互作用对于理解细胞过程至关重要。我们介绍了将氟动态核极化（DNP）与快速魔角旋转（MAS）核磁共振（NMR）光谱相结合，作为一种研究哺乳动物细胞中蛋白质的强大技术。我们以严重急性呼吸综合征冠状病毒2 5F-Trp-N蛋白为例展示了我们的方法，该蛋白通过电穿孔导入人类细胞。观察到DNP信号增强了30至40倍，这意味着实验时间节省了1000多倍。在几分钟内就获得了细胞中纳摩尔量蛋白质的高信噪比光谱。获得了具有显著灵敏度和分辨率的二维F-F偶极相关光谱，展示了相距约10埃的氟原子相关的F-F交叉峰。这项工作为基于氟DNP增强的MAS NMR在细胞中探测蛋白质结构、动力学和配体相互作用的应用铺平了道路。
