The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein’s backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone ¹H, ¹³C, and ¹⁵N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, ¹⁵N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the “missing” amide resonances for N96-L106 in the ¹H-¹⁵N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to μs timescale and/or is heterogenous. These “missing” amide resonances do not unambiguously appear in the ¹H-¹⁵N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to μs timescale dynamics is the physical basis for a previously suggested “fluidity” of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.

Betacoronavirus SARS-CoV-2 非结构蛋白 Nsp9 是一种含有 113 个残基的蛋白，对病毒复制至关重要，因此是开发针对 COVID19 感染的疗法的潜在目标。为了深入了解溶液中蛋白质骨架的动力学，并通过化学位移扰动研究加速配体结合表面的识别和绘图，Nsp9 的骨架¹ H、¹³ C 和¹⁵ N NMR 化学位移已被广泛分配。这些任务通过制备约 70% 的氘化样品和残留物特异性¹⁵N 标记的样本（V、L、M、F 和 K）。分配的一个主要特征是 N96-L106 在¹ H- ¹⁵ N HSQC 光谱中的“缺失”酰胺共振，该区域包含几乎完整的 C 末端 α-螺旋，形成同源二聚体界面的主要部分SARS-CoV-2 Nsp9 的晶体结构，表明该区域要么在 ms 到 μs 时间尺度内经历中间运动和/或是异质的。这些“缺失”的酰胺共振并没有明确地出现在¹ H- ¹⁵以 0.0007 mM 的浓度收集的 SARS-CoV-2 Nsp9 的 N HSQC 谱。在此浓度下，在检测限下，天然质谱表明该蛋白质完全处于单体状态，这表明 Nsp9 C 末端的中间运动可能是由于分子内动力学。也许这种中间的 ms 到 μs 时间尺度动力学是先前提出的 C 末端螺旋的“流动性”的物理基础，这可能是同嗜性 (Nsp9-Nsp9) 和假定的异嗜性 (Nsp9-未知) 蛋白质-蛋白质相互作用的原因。