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

基本信息

批准号：
10253719
负责人：
Adriaan Bax
金额：
$ 99.52万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10253719
关键词：
2019-nCoV 3-Dimensional Acidosis Active Sites Algorithms Area Biological Cellular Stress Chemicals Collection Core Protein Coupling Data Data Analyses Databases Diffusion Dimerization Dioxanes Disease Dissociation Environmental Risk Factor Exhibits Exposure to Four-dimensional Friction Goals HSPB1 gene Heat shock proteins Heat-Shock Response Human Hydrophobic Surfaces Hydrophobicity Hydrostatic Pressure Length Life Ligands Measurement Measures Methods Modeling Molecular Chaperones Molecular Conformation Molecular Structure Motion NMR Spectroscopy Pattern Peptide Hydrolases Physiologic pulse Protein Dynamics Proteins Radial Reference Standards Relaxation Reporter Residual state Roentgen Rays Rotation Sampling Series Solvents Structure Surface Time Ubiquitin Urea Vertebral column Virus Viscosity Water alpha synuclein alpha-Crystallins combat data acquisition dimer experimental study monomer mutant novel polypeptide pressure prevent protein aggregation protein folding protein structure restraint small molecule

项目摘要

For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. However, while the impact of pressure on the viscosity of water is well known, we find that elevated pressures increase the Rh of dioxane, a commonly used reference standard, and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction observed by 13C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurements of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure and as a function of urea concentration show that Rh values of both the folded and the unfolded states remain nearly invariant. At ca 23 angstrom, the Rh of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) alpha-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of both unfolded ubiquitin and alpha-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions. Small heat-shock proteins (sHSPs) are molecular chaperones that respond to cellular stresses to combat protein aggregation. HSP27 is a critical human sHSP that forms large, dynamic oligomers whose quaternary structures and chaperone activities depend on environmental factors. Upon exposure to cellular stresses, such as heat shock or acidosis, HSP27 oligomers can dissociate into dimers and monomers, which leads to significantly enhanced chaperone activity. The structured core of the protein, the alpha-crystallin domain (ACD), forms dimers and can prevent the aggregation of substrate proteins to a similar degree as does the full-length protein. We have shown that when the ACD dimer dissociates into monomers, it partially unfolds and exhibits enhanced activity. Using solution-state NMR spectroscopy to characterize the structure and dynamics of the HSP27 ACD monomer, we have shown that the monomer is stabilized at low pH and that its backbone chemical shifts, N-15 relaxation rates, and 1H-15N residual dipolar couplings suggest structural changes and rapid motions in the region responsible for dimerization. By analyzing the solvent accessible and buried surface areas of sHSP structures in the context of a database of dimers that are known to dissociate into disordered monomers, we predict that ACD dimers from sHSPs across all kingdoms of life may partially unfold upon dissociation. This resulted in a general model in which conditional disorder-the partial unfolding of ACDs upon monomerization-is a common mechanism for sHSP activity. A series of novel three- and four-dimensional NMR experiments has been developed that provides access to the structure and dynamics of the 608-residue homodimeric Main protease of the SARS-CoV-2 virus. This protein is larger than what is readily accessible to the standard NMR approaches used, but the novel methods combined with 900 MHz high-field measurements provided sufficient data for a detailed structural analysis which reveals subtle but statistically very significant differences relative to all X-ray structures available so far. A characterization of the backbone dynamics shows a strong effect of ligands on the dynamics of the protein backbone in the active site region.

对于未折叠的蛋白质，Rh 提供了关于整体平均构象和多肽链扩展程度（作为添加变性剂的函数）的敏感报告。静水压是化学变性剂的一种方便且可逆的替代品，可用于研究蛋白质折叠，并且可以在单个样品上进行 NMR 测量。脉冲场梯度核磁共振波谱广泛用于测量溶液中生物分子的平移扩散和流体动力学半径 (Rh)。然而，虽然压力对水粘度的影响是众所周知的，但我们发现，升高的压力会增加二恶烷（一种常用的参考标准）和其他小分子的 Rh，其量与其疏水性相关，同时旋转力也会相应增加。通过 13C 纵向松弛时间观察到的摩擦力。这些数据表明疏水表面在高压下与水的耦合更紧密。对压力敏化泛素突变体 (VA2-泛素) 的未折叠状态作为静水压力和尿素浓度的函数的平移扩散测量表明，折叠状态和未折叠状态的 Rh 值几乎保持不变。在大约 23 埃时，完全压力变性状态的 Rh 基本上与尿素变性状态没有区别，并且接近 76 个残基的理想化无规卷曲的预期值。本质无序蛋白 (IDP) α-突触核蛋白在高于 2 kbar 的压力下表现出轻微的压缩。未折叠的泛素和α-突触核蛋白的扩散均受到样品浓度的显着影响，表明定量测量需要在稀释条件下进行。小热休克蛋白 (sHSP) 是分子伴侣，可响应细胞应激以对抗蛋白质聚集。 HSP27 是一种关键的人类 sHSP，可形成大型动态寡聚物，其四级结构和伴侣活性取决于环境因素。当暴露于热休克或酸中毒等细胞应激时，HSP27 寡聚体可以解离成二聚体和单体，从而导致伴侣活性显着增强。该蛋白质的结构核心，α-晶状体蛋白结构域 (ACD)，形成二聚体，并且可以防止底物蛋白质的聚集，其程度与全长蛋白质相似。我们已经证明，当 ACD 二聚体解离成单体时，它会部分展开并表现出增强的活性。使用溶液态核磁共振波谱来表征 HSP27 ACD 单体的结构和动力学，我们发现该单体在低 pH 下稳定，并且其主链化学位移、N-15 弛豫速率和 1H-15N 残余偶极耦合表明导致二聚化的区域的结构变化和快速运动。通过在已知解离成无序单体的二聚体数据库中分析 sHSP 结构的溶剂可及和埋藏表面积，我们预测来自所有生命王国的 sHSP 的 ACD 二聚体可能在解离后部分展开。这产生了一个通用模型，其中条件障碍（单体化时 ACD 的部分展开）是 sHSP 活性的常见机制。一系列新颖的三维和四维核磁共振实验已经开发出来，可以了解 SARS-CoV-2 病毒的 608 个残基同二聚体主要蛋白酶的结构和动力学。这种蛋白质比使用的标准 NMR 方法容易获得的蛋白质要大，但结合 900 MHz 高场测量的新颖方法为详细的结构分析提供了足够的数据，揭示了相对于所有 X 射线的微妙但统计上非常显着的差异迄今为止可用的结构。主链动力学的表征表明配体对活性位点区域中蛋白质主链的动力学有很强的影响。