Methods to accelerate protein structure determination by solution NMR

通过溶液核磁共振加速蛋白质结构测定的方法

• 批准号: 10697720
• 负责人: Adriaan Bax
• 金额: $ 65.87万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697720
• 关键词: 2019-nCoV; 3-Dimensional; Active Sites; Agreement; Algorithms; Binding; Chemicals; Collection; Data; Data Analyses; Data Set; Databases; Deposition; Detection; Equilibrium; Four-dimensional; Goals; Hydrogen; Hydrogen Bonding; Measurement; Measures; Meta-Analysis; Methods; Modeling; Molecular Conformation; Molecular Structure; Motion; NMR Spectroscopy; Pattern; Peptide Hydrolases; Population; Positioning Attribute; Process; Protein Dynamics; Proteins; Protons; Residual state; Resolution; Roentgen Rays; SARS-CoV-2 protease; Sampling; Scheme; Spectrum Analysis; Structure; Sulfhydryl Compounds; System; Time; Vertebral column; Virus; Work; X ray diffraction analysis; data acquisition; experimental study; falls; improved; novel; polypeptide; protein data bank; protein structure; quantum; restraint

Structural analysis of meta-stable proteins is challenging because measured parameters often reflect a population weighted average over the different states dynamically sampled by such systems. New three- and four-dimensional NMR experiments have been developed that provide access to the structure and dynamics of systems that are large in comparison to proteins normally studied by NMR spectroscopy, such as the homodimeric Main protease of the SARS-CoV-2 virus. The novel methods combined with 900 MHz high-field measurements provided sufficient data for a detailed structural characterization that reveals subtle but statistically quite significant differences relative to all X-ray structures available so far. A characterization of the backbone motions shows a strong effect of substrate binding on the dynamics of the protein backbone in the active site region. Specifically, new schemes to acquire and process triple-resonance data for measurement of residual dipolar couplings (RDCs) in combination with non-uniform sampling have been developed that increase the precision at which such RDCs can be extracted from the data. The TROSY-HNCO 3D NMR experiments was extended in a manner similar to that of the original amplitude-modulated RDC TROSY Spectroscopy (ARTSY) experiment to obtain vastly improved spectral resolution, which allowed the measurement of a nearly complete set of data for the SARS-CoV-2 main protease, and to assess its structure in solution and to compare it with the many X-ray structures deposited in the protein databank (PDB). Results show that although, on average, solution measurements are in good agreement with the X-ray data, in several localized regions the solution data fall outside the range observed in the nearly 200 X-ray structures. Substantial differences appear in the active site region, and refinement of the solution NMR structure of this homodimeric structure indicates that many of the residues near the active site exist in a microsecond timescale equilibrium between multiple states. None of the X-ray structures, provides a good fit to the solution NMR RDCs, although some improvement in agreement is observed when comparing the NMR data to ensemble models developed by the PHENIX method to fit X-ray diffraction data to ensembles of structures. In related work, we have developed methods that permit NMR detection of sulfhydryl resonances in proteins. Ten out of 11 Cys thiol resonances proved observable, but their chemical shifts mostly disagreed with those of AlphaFold2 models, indicating that the proton placement in such models remains far from optimal. Good agreement with experimental chemical shifts is observed when using quantum-chemical calculation to define the energetic minima for the positions of these hydrogen atoms, revealing substantially different hydrogen bonding networks.

亚稳定蛋白质的结构分析具有挑战性，因为测量的参数通常反映此类系统动态采样的不同状态的群体加权平均值。 新的三维和四维核磁共振实验已经开发出来，可以了解与核磁共振波谱通常研究的蛋白质相比较大的系统的结构和动力学，例如 SARS-CoV-2 病毒的同二聚体主要蛋白酶。 这种新颖的方法与 900 MHz 高场测量相结合，为详细的结构表征提供了足够的数据，揭示了相对于迄今为止所有可用 X 射线结构的微妙但统计上相当显着的差异。 主链运动的表征显示底物结合对活性位点区域中蛋白质主链的动力学有强烈影响。 具体来说，已经开发了结合非均匀采样来采集和处理三重共振数据以测量残余偶极耦合 (RDC) 的新方案，提高了从数据中提取此类 RDC 的精度。 TROSY-HNCO 3D NMR 实验以类似于原始调幅 RDC TROSY 光谱 (ARTSY) 实验的方式进行了扩展，以获得大幅提高的光谱分辨率，从而可以测量 SARS 的近乎完整的数据集。 CoV-2 主要蛋白酶，并评估其在溶液中的结构，并将其与蛋白质数据库 (PDB) 中存储的许多 X 射线结构进行比较。 结果表明，虽然平均而言，解测量结果与 X 射线数据非常一致，但在几个局部区域中，解数据超出了近 200 个 X 射线结构中观察到的范围。 活性位点区域出现显着差异，并且对该同二聚体结构的溶液核磁共振结构的细化表明，活性位点附近的许多残基存在于多种状态之间的微秒时间尺度平衡中。 尽管在将 NMR 数据与 PHENIX 方法开发的系综模型进行比较以将 X 射线衍射数据拟合到结构系综时观察到一致性有所改善，但没有一个 X 射线结构能够很好地拟合解 NMR RDC。 在相关工作中，我们开发了允许 NMR 检测蛋白质中的巯基共振的方法。 11 个 Cys 硫醇共振中有 10 个被证明是可观察到的，但它们的化学位移大多与 AlphaFold2 模型的化学位移不一致，这表明此类模型中的质子位置仍然远非最佳。 当使用量子化学计算来定义这些氢原子位置的能量最小值时，观察到与实验化学位移的良好一致性，揭示了本质上不同的氢键网络。