NMR--New Methods And Molecular Structure Determination

我们已经扩展了我们的技术，用于研究弱排列条件下溶液中的大分子结构。新的进展集中在精确测量质子之间的偶极相互作用上。我们已经证明，在有利的条件下，这种直接相互作用可以在长达12？的距离内观察到，而不需要特殊的同位素浓缩。1H-1H偶极相互作用既取决于核间距离，也取决于核间载体相对于分子排列框架的取向，从而构成了对广泛使用的仅取决于距离的1H-1H NOE相互作用的直接补充。1H-1H偶极相互作用和1H-13C偶联的组合被证明足以准确地定义DNA寡聚体的溶液结构，但也表明特别是嘧啶核苷酸的脱氧核糖单元与单一的静态构象不相容。然而，它们与糖在C2？-Endo构象(主要形式)和次要C3？-Endo形式之间快速切换的模型是一致的。我们最新开发的实验允许精确测量每个结构单元(核糖、脱氧核糖或氨基酸)前所未有的偶极偶联，从而提供了一种新的途径来探索每个单元单独经历的对准程度，从而提供关于这些单元相对于大分子整体框架的移动性或缺乏程度的直接信息。 前人的工作是根据观察到的磁化率各向异性导致的磁排向程度和实际观察到的排列来探讨核酸的分子内动力学问题。这通常会导致得出结论，即核酸中存在非常大的内部动力学和铰链运动。我们用量子计算和实验方法重新分析了核酸碱基的本征磁化率，发现比其他人通常使用的值要小得多。有了我们的新碱基磁化率值，核酸中结构域运动的幅度比以前得出的结论要小得多。这一发现对于理解蛋白质-核酸识别中的热力学成分具有直接的相关性。

We have extended our technology for studying macromolecular structure in solution under weakly aligning conditions. New developments focus on the accurate measurement of dipolar interactions between protons. We have demonstrated that under favorable conditions, such direct interactions are observable over distances of up to 12 ?, without requiring special isotopic enrichment. The 1H-1H dipolar interaction depends on both the internuclear distance and on the orientation of the internuclear vector relative to the alignment frame of the molecule, and thereby constitutes a direct complement to the widely used 1H-1H NOE interaction, which depends only on the distance. The combination of 1H-1H dipolar interactions and 1H-13C couplings was shown to be sufficient to accurately define the solution structure of a DNA oligomer, but also showed that in particular the deoxyribose units of pyrimidine nucleotides were not compatible with a single, static conformation. They are, however, in agreement with a model where the sugar rapidly switches between a C2?-endo conformer (the dominant form), and a minor C3?-endo form. Our newly developed experiments permit the accurate measurement of an unprecedented number of dipolar couplings per structural unit (ribose, deoxyribose, or amino acid) and thereby provides a new avenue to explore the degree of alignment each unit experiences individually, thereby providing direct information on the degree of mobility, or lack thereof, of such units relative to the overall frame of the macromolecule. Previous work by others has explored the issue of intramolecular dynamics in nucleic acids on the basis of the observed degree of magnetic alignment, resulting from magnetic susceptibility anisotropy, and the actually observed alignment. Frequently, this led to conclusions that very large amplitude internal dynamics and hinge motions were present in nucleic acids. We have re-analyzed the intrinsic magnetic susceptibility of nucleic acids bases by both quantum-computational and experimental methods and find considerably smaller values than were commonly used by others. With our new base susceptibility values, the amplitude of domain motions in nucleic acids is of considerably smaller amplitude than concluded previously. This finding is of direct relevance for understanding the thermodynamic components in protein-nucleic acid recognition.