NMR--New Methods And Molecular Structure Determination

我们扩展了在弱对准条件下研究溶液中大分子结构的技术。新的发展集中在质子间偶极相互作用的精确测量上。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 ？，不需要特殊的同位素富集。1H-1H偶极相互作用既取决于核间距离，也取决于核间矢量相对于分子排列框架的取向，因此构成了广泛使用的1H-1H NOE相互作用的直接补充，后者仅取决于距离。1H-1H偶极相互作用和1H-13C偶联的结合被证明足以准确地定义DNA寡聚物的溶液结构，但也表明特别是嘧啶核苷酸的脱氧核糖单元不兼容单一的静态构象。然而，它们与一个模型是一致的，即糖在C2？-末端的构象（主要形式）和一个次要的C3？endo形式。我们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）中前所未有数量的偶极偶联，从而为探索每个单元单独经历的排列程度提供了新的途径，从而提供了有关这些单元相对于大分子整体框架的迁移程度或缺乏迁移程度的直接信息。

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.