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SAUPE基质包含五个独立元素，从而可以产生多达五个线性独立的比对条件。 在这些条件下，NMR光谱法测量了核内残留偶性耦合（RDC），在五维对齐空间中正交，可访问幅度，不对称和核心内部载体运动的方向。我们先前证明了小蛋白质结构域GB3（56个残基），可以通过产生一系列对域的域骨架结构可忽略的保守性突变体，从而在PF1噬菌体的单个液体晶体介质中适当地进行正交对准条件。 突变涉及几个暴露溶剂的侧技术的电荷，以及通常用于蛋白质纯化的N-或C末端HIS-TAG肽对蛋白质的延伸。 这些蛋白质突变体绘制了五维对齐空间，为结构和动力学提供了独特的见解，并提供了对各向异性参数的访问，例如13C，15N和1H化学屏蔽量张量。 现在已经开发了这项技术，以便为GB3中有序的骨干酰胺部分推导特定地点的1H化学位移各向异性（CSA）张量。实验输入数据包括残留的化学移动各向异性（RCSA），在六个突变体中测量，这些突变体在溶解在液晶PF1悬浮液中时，相对于静态磁场的对齐相对于静态磁场的一致性不同，并在1H（N）CSA Tensor和1H-15N和1H-15N，1H-13C'或1H-1H-1H-1H-1H-1H-1HC（1H）CSA张量之间的交叉相关放松速率（通过假设1H（n）CSA张量相对于肽平面（三参数拟合）或没有这种前提（五参数拟合）的结果是对称的，从而得出非常相似的结果，从而证实了实验输入数据的鲁棒性，并且可以使良好的近似近似近似近似orthers Ortherals Ortherals或Pettide ofteCtideSpeptideSpeptideSpeptideSpeptideSpeptideSpeptide。发现1H（n）CSA张量与轴向对称性有很强的偏差，最屏蔽的张量分量大致平行于N-H载体，而最小屏蔽的组分与肽平面正交。从GB3 X射线结构中采集的H键几何形状中的N-甲基乙酰酰胺和乙酰酰胺成对上的DFT计算与实验数据相关，并表明H键作用占1H（n）CSA中的h键作用主导的变化。使用实验得出的1H（n）CSA张量，在Ca 1200 MHz处发现最窄的1H（N）Trosy线宽度所需的最佳弛豫干扰效果。