Protein and nucleic acid structure and dynamics from residual dipolar couplings

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。此外，我们还证明了抗病毒和抗细菌分子角鲨胺可以采用适合蛋白质比对的液晶相。蛋白质泛素相对较强的比对使得其他人关于在缓慢(&gt；10 ns)时间尺度上存在大的脊椎运动的结论被重新处理。我们在角鲨胺介质中新测量的RDC在很大程度上与文献结论不一致，并指出蛋白质二级结构区域的运动范围要窄得多，而对于环区，数据与结晶学研究中观察到的构象集合很好地吻合。 我们已经开发和演示了新的方法来获得对应于纯正交排列张量的实验数据集，从而消除了在各种排列条件下测量的RDC值之间的内在相关性。血凝素融合肽的应用为了解其结构和内部动力学提供了独特的见解。

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. Moreover, we have demonstrated that the anti-viral and anti-bacterial molecule squalamine can adopt a liquid crystalline phase suitable for protein alignment. Relatively strong alignment of the protein ubiquitin allowed the conclusion by others regarding the presence of large backbone motions on a slow (>10 ns) time scale to be re-addressed. Our newly measured RDCs in squalamine medium are largely inconsistent with the literature conclusions and point to a much narrower range of motions for regions of the protein engaged in secondary structure, whereas for loop regions the data fit well to the ensemble of conformations observed in crystallographic studies. We have developed and demonstrated new methods for obtaining experimental data sets that correspond to purely orthogonal alignment tensors, thereby removing the intrinsic correlation present between RDC values measured under a variety of alignment conditions. Application to the fusion peptide of hemagglutinin provided unique insights into its structure and internal dynamics.