Protein and nucleic acid structure and dynamics from residual dipolar couplings

描述蛋白质在液晶介质中的排列的Saupe矩阵包含五个独立的元素，使得能够产生多达五个线性独立的排列条件。 在这些条件下，在五维排列空间中正交的NMR光谱法测量核间残余偶极耦合（RDC），提供了对核间矢量运动的幅度、不对称性和方向的访问。我们先前证明了对于小蛋白质结构域GB 3（56个残基），通过产生一系列对结构域的时间平均骨架结构具有可忽略的影响的保守突变体，可以在Pf 1噬菌体的单一液晶介质中产生适当的正交对齐条件。 突变涉及几个溶剂暴露侧链的电荷变化，以及通过N-或C-末端His-标签肽（通常用于蛋白质纯化）的蛋白质延伸。 这些蛋白质突变体绘制了五维对齐空间，提供了对结构和动力学的独特见解，并提供了对各向异性参数的访问，如13 C，15 N和1H化学屏蔽张量。 这项技术现在已经得到进一步发展，以获得特定位点的1H化学位移各向异性（CSA）张量的有序骨架酰胺部分GB 3。实验输入数据包括残余化学位移各向异性（RCSA），在溶解于液晶Pf 1悬浮液中时相对于静磁场不同排列的六种突变体中测量，以及1H（N）CSA张量与1H-15 N、1H-13 C '或1H-13 C（α）偶极相互作用之间的交叉相关弛豫速率。假设1H（N）CSA张量相对于肽平面对称（三参数拟合）或不假设此前提（五参数拟合）的分析产生非常相似的结果，证实了实验输入数据的鲁棒性，并且在良好的近似下，其中一个主成分与肽平面正交。1H（N）CSA张量被发现强烈偏离轴对称，最屏蔽的张量分量大致平行于N-H矢量，最少屏蔽的分量垂直于肽平面。对GB 3 X射线结构中的N-甲基乙酰胺和乙酰胺氢键几何构型的DFT计算与实验数据相关，并表明氢键效应主导1H（N）CSA的变化。利用实验得到的1H（N）CSA张量，在约1200 MHz处找到了最宽1H（N）TROSY线宽所需的最佳弛豫干涉效应。

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.