Kumar Tekwani Movellan, Wenkai Zhu, Daniel Banks, James Kempf, Brent Runge, Angela M. Gronenborn, Tatyana Polenova
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引用次数: 0
摘要
获取细胞内成分的原子级信息是结构生物学的一个重点。阐明蛋白质的特定结构和动态特征及其在细胞环境中的相互作用对于理解细胞过程至关重要。我们介绍了 19 F 动态核偏振(DNP)与快速魔角旋转(MAS)核磁共振(NMR)光谱相结合的强大技术,用于研究哺乳动物细胞中的蛋白质。我们在电穿孔到人体细胞中的严重急性呼吸系统综合征冠状病毒 2 5F-Trp-N NTD 蛋白上演示了我们的方法。观察到 DNP 信号增强了 30 到 40 倍,节省了超过 1000 倍的实验时间。在几分钟内就能获得细胞中纳摩尔量蛋白质的高信噪比光谱。获得的二维 19 F- 19 F 双极相关光谱具有极高的灵敏度和分辨率,显示出与相距约 10 埃的氟原子相关的 19 F- 19 F 交叉峰。这项工作为在细胞中应用 19 F DNP 增强 MAS NMR 探测蛋白质结构、动力学和配体相互作用铺平了道路。
Expanding the tool box for native structural biology: 19 F dynamic nuclear polarization with fast magic angle spinning
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 19 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 NTD 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 19 F- 19 F dipolar correlation spectra with remarkable sensitivity and resolution were obtained, exhibiting 19 F- 19 F cross peaks associated with fluorine atoms as far as ~10 angstroms apart. This work paves the way for 19 F DNP-enhanced MAS NMR applications in cells for probing protein structure, dynamics, and ligand interactions.
期刊介绍:
Science Advances, an open-access journal by AAAS, publishes impactful research in diverse scientific areas. It aims for fair, fast, and expert peer review, providing freely accessible research to readers. Led by distinguished scientists, the journal supports AAAS's mission by extending Science magazine's capacity to identify and promote significant advances. Evolving digital publishing technologies play a crucial role in advancing AAAS's global mission for science communication and benefitting humankind.