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: 8349704
• 负责人: Ad Bax
• 金额: $ 29.99万
• 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/8349704
• 关键词: Acetamides; Amides; Anisotropy; Bacteriophage Pf1; C-terminal; Charge; Chemicals; Data; Elements; Generations; Hour; Hydrogen Bonding; Liquid substance; Maps; Measurement; Measures; Methods; Motion; Mutation; NMR Spectroscopy; Peptides; Process; Proteins; Relative (related person); Relaxation; Residual state; Roentgen Rays; Series; Site; Solutions; Solvents; Structure; Suspension substance; Suspensions; System; Techniques; Technology; Tertiary Protein Structure; Time; Variant; Vertebral column; Width; conformer; insight; magnetic field; mutant; nucleic acid structure; protein purification; 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. This technology has now been developed further in order to derive site-specific 1H chemical shift anisotropy (CSA) tensors for the well-ordered backbone amide moieties in GB3. Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the 1H(N) CSA tensor and either the 1H-15N, the 1H-13C', or the 1H-13C(alpha) dipolar interactions. Analyses with the assumption that the 1H(N) CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. 1H(N) CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N-H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of N-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the 1H(N) CSA. Using experimentally derived 1H(N) CSA tensors, the optimal relaxation interference effect needed for narrowest 1H(N) TROSY line widths is found at ca 1200 MHz.

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。 这项技术现在已经得到了进一步的发展，以便得到GB3中有序的主链酰胺部分的特定位置的1H化学位移各向异性(CSA)张量。实验输入数据包括残余化学位移各向异性(RCSA)，在六个突变体中测量，当溶解在液晶PF1悬浮液中时，这些突变体相对于静态磁场的排列方式不同，以及1H(N)CSA张量与1H-15N、1H-13C‘或1H-13C(α)偶极相互作用之间的交叉关联驰豫速率。在假设1H(N)CSA张量相对于多肽平面对称(三参数拟合)或没有这一前提(五参数拟合)的情况下进行分析，得到非常相似的结果，证实了实验输入数据的稳健性，并且在良好的近似值下，其中一个主成分取向与多肽平面垂直。发现1H(N)CSA张量强烈偏离轴对称性，屏蔽最多的张量分量大致平行于N-H矢量，最小屏蔽分量与肽平面垂直。对从GB3X-射线结构获得的氢键几何构型中N-甲基乙酰胺和乙酰胺对的密度泛函计算与实验数据相关联，表明氢键效应主导了1H(N)CSA的变化。利用实验得到的1H(N)CSA张量，在约1200 MHz处找到了最窄的1H(N)TROSY线宽所需的最佳弛豫干涉效应。