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，能够产生多达五个线性独立的排列条件。在这些条件下，通过核磁共振波谱测量核间残余偶极耦合（RDCs），在五维对准空间中正交，提供了核间矢量运动的幅度，不对称性和方向。我们之前证明了小蛋白结构域GB3（56个残基）可以在Pf1噬菌体的单一液晶培养基中产生适当的正交对准条件，通过产生一系列保守突变体，这些突变体对该结构域的时间平均骨干结构的影响可以忽略不计。突变包括几种暴露于溶剂的侧链电荷的变化，以及通常用于蛋白质纯化的N端或c端his标签肽对蛋白质的延伸。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，并提供了对各向异性参数（如13C， 15N和1H化学屏蔽张量）的访问。

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. For base-paired nucleic acids, we find that imino N-H vector orientations can deviate considerably from idealized geometry, and agree considerably better with results from DFT calculations that with idealized geometry.