Protein and nucleic acid structure and dynamics from residual dipolar couplings

残余偶极耦合的蛋白质和核酸结构和动力学

• 批准号: 9357208
• 负责人: Ad Bax
• 金额: $ 45.14万
• 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/9357208
• 关键词: Amyloid beta-Protein; Bacteriophages; Benchmarking; C-terminal; Charge; Chemicals; Data; Detergents; Development; Disease; Elements; Equation; Equilibrium; Generations; Hour; Lipids; Liquid substance; Maps; Measurement; Methods; Modeling; Motion; Mutation; NMR Spectroscopy; Peptides; Plant Roots; Population; Process; Proteins; Protocols documentation; Relaxation; Residual state; Roentgen Rays; Sampling; Series; Solvents; Structure; System; Technology; Tertiary Protein Structure; Time; Vertebral column; alpha synuclein; conformer; improved; insight; molecular dynamics; mutant; nucleic acid structure; protein folding; protein purification; restraint; vector

The Saupe matrix describing protein alignment in a liquid crystalline medium contains five independent elements, enabling the generation of up to five linearly independent alignment conditions. Measurement of internuclear residual dipolar couplings (RDCs) by NMR spectroscopy under these conditions, orthogonal in five-dimensional alignment space, provides access to the amplitude, asymmetry, and direction of motions of the internuclear vector. We previously demonstrated for the small protein domain GB3 (56 residues) that suitably orthogonal alignment conditions can be generated in a single liquid crystalline medium of Pf1 phage, by generating a series of conservative mutants that have negligible impact on the time-averaged backbone structure of the domain. Mutations involve changes in the charge of several solvent-exposed sidechains, as well as extension of the protein by either an N- or C-terminal His-tag peptide, commonly used for protein purification. These protein mutants map out the five-dimensional alignment space, providing unique insights into the structure and dynamics, and providing access to anisotropic parameters such as the 13C, 15N and 1H chemical shielding tensors. Rather than modifying the charge distribution to alter protein alignment, we have demonstrated that for detergent-solubilized systems it is also possible to change alignment by altering the detergent and lipid composition of the sample. Combining these technologies, we have engaged in a modelfree study of the sidechain conformational distributions in a small model protein, GB3. We find that the RDC data are compatible with a single narrow distribution of sidechain chi1 angles for only about 40% of the residues. For more than half of the residues, populations greater than 10% for a second rotamer are observed, and four residues require sampling of three rotameric states to fit the RDC data. In virtually all cases, sampled chi11 values are found to center closely around ideal g-, g+ and t rotameric angles, even though no rotamer restraint is used when deriving the sampled angles. The root-mean-square difference between experimental 3JHH couplings and those predicted by the Haasnoot-parameterized, motion-adjusted Karplus equation reduces from 2.05 Hz to 0.75 Hz when using the new rotamer analysis instead of the 1.1- X-ray structure as input for the dihedral angles. A comparison between observed and predicted 3JHH values suggests that the root-mean-square amplitude of chi1 angle fluctuations within a given rotamer well is ca 20. The quantitatively defined sidechain rotamer equilibria obtained from our study set new benchmarks for evaluating improved molecular dynamics force fields, and also will enable further development of quantitative relations between sidechain chemical shift and structure. In a separate but related approach, we use the measurement of multiple three-bond J couplings as a new method to define the root-mean-square amplitude of backbone phi angle fluctuations. Although for most folded proteins, these fluctuations are too small (<15 deg) to be detectable by this method, for intrinsically disordered proteins an accurate, residue-specific estimate can be made. This approach has been used to study two amyloidogenic proteins, alpha-synuclein and Abeta peptides.

描述液晶介质中蛋白质比对的索普矩阵包含五个独立的元素，从而能够产生最多五个线性独立的比对条件。在这些条件下，用核磁共振波谱测量核间残留偶极耦合(RDC)，在五维排列空间中正交，提供了核间矢量的幅度、不对称性和运动方向的途径。我们先前证明了对于小的蛋白质结构域GB3(56个残基)，通过产生一系列保守的突变体，可以在PF1噬菌体的单一液晶介质中产生合适的正交比对条件，这些突变体对结构域的时间平均骨架结构的影响可以忽略不计。突变包括几个暴露在溶剂中的侧链电荷的变化，以及蛋白质的N-末端或C-末端组氨酸标记肽的延伸，通常用于蛋白质纯化。这些蛋白质突变体绘制了五维排列空间，提供了对结构和动力学的独特见解，并提供了访问各向异性参数的途径，如13C，15N和1H化学屏蔽张量。 我们没有改变电荷分布来改变蛋白质的排列，而是证明了对于洗涤剂溶解的系统，也可以通过改变样品的洗涤剂和脂肪组成来改变排列。结合这些技术，我们对小模型蛋白GB3中的侧链构象分布进行了非模型研究。我们发现，对于大约40%的残基，RDC数据与单一狭窄的侧链Chi1角分布是兼容的。对于超过一半的残基，观察到第二个旋转体的群体大于10%，其中四个残基需要对三个旋转态进行采样才能符合RDC数据。在几乎所有情况下，采样的Chi11值都紧密围绕理想的g-、g+和t旋转角，即使在推导采样角度时没有使用旋转限制。当使用新的旋量分析代替1.1X-射线结构作为二面角的输入时，实验3JHH耦合与Haasnoot参数、运动调整的Karplus方程预测的3JHH耦合之间的均方根差从2.05 Hz减小到0.75 Hz。3JHH值的实测值和预测值的比较表明，在给定的转子势垒内，Chi1角波动的均方根幅度约为20。从我们的研究中获得的定量定义的侧链转子平衡为评估改进的分子动力学力场提供了新的基准，也将使侧链化学位移与结构之间的定量关系得到进一步发展。 在另一种独立但相关的方法中，我们使用多个三键J耦合的测量作为一种新的方法来定义主链Phi角波动的均方根幅度。虽然对于大多数折叠蛋白质来说，这些波动太小(&lt；15℃)，这种方法无法检测到，但对于内在无序的蛋白质，可以做出准确的、特定残基的估计。这种方法已经被用来研究两种淀粉样蛋白，α-突触核蛋白和Aβ多肽。