NMR STUDIES OF BIOMOLECULAR STRUCTURE, FUNCTION, AND DYNAMICS

本项目的目标是：(1)开发和修改核磁共振方法 对于共振的分配和结构的分析，以及 溶液中分子的动力学，以及(2)将这些方法应用于 与环境健康有关的令人关注的问题。在过去 去年，该项目的主要重点一直是开发 转移NOE(TRNOE)技术及其在生产中的应用 核苷类药物结节菌素的构象测定 与大肠杆菌嘌呤核苷磷酸化酶(PNPase)的复合体。几个 建立了分析结合动力学和解离速率的方法 2 400 S-1和12 0 0 S-1、2 0xC和10xC的常数分别为 下定决心。缓蚀剂2‘位的特定氢化 为了减少或消除几个，已经进行了杀结核 缓蚀剂中重要的自旋扩散途径。没想到，被绑住了 结节杀菌素被发现对应于两种截然不同的构象： 一个主要的同步构象和一个次要的反构象。配基 进一步涉及杀菌素和9-去氮胞苷的竞争研究 认为这两种绑定模式都涉及到与活跃的 酶的位置。其他研究一直在继续探索 苯硼酸衍生物与丝氨酸的相互作用 枯草杆菌蛋白水解酶和模型配体。除了这些之外， 大分子研究--编辑核磁共振氢谱的新途径 选择性地基于标量偶合碳-13核的位移 已在几个模型系统中进行了评估和应用。质子核磁共振研究 还进行了皮质酮和皮质酮的结构测定 细胞色素突变产生的脱氧皮质酮代谢物 P450。一项涉及使用核磁共振波谱研究的新举措 人类雌激素受体的结构和构象方面 最近开始的。

The aims of this project are: (1) to develop and modify NMR methodologies for the assignment of resonances and the analysis of the structure and dynamics of molecules in solution, and (2) to apply these methods to problems of concern in relation to environmental health. During the past year, the main emphasis of this project has been on the development of the transferred NOE (TRNOE) technique and its application to the determination of the conformation of the nucleoside drug tubercidin in the complex with E. coli purine nucleoside phosphorylase (PNPase). Several methods were developed to analyze binding kinetics, and dissociation rate constants of 2400 s-1 and 1200 s-1 and 20xC and 10xC, respectively, were determined. Specific deuteration of the 2' position of the inhibitor tubercidin has been carried out in order to reduce or eliminate several important spin diffusion pathways in the inhibitor. Unexpectedly, bound tubercidin was found to correspond to two very different conformations: a major syn conformation and a minor, anticonformation. Ligand competition studies involving tubercidin and 9-deazainosine further suggest that both modes of binding involve interaction with the active sites of the enzyme. Other studies have continued to probe the interaction of benzeneboronic acid (BBA) derivatives with the serine protease subtilisin and with model ligands. In addition to these macromolecular studies, a new approach to editing 1H NMR spectra selectively based on the shifts of scalar coupled carbon-13 nuclei has been evaluated and applied to several model systems. Proton NMR studies were also carried out to determine the structure of corticosterone and deoxycorticosterone metabolites produced by mutated forms of cytochrome P450. A new initiative involving the use of NMR spectroscopy to study structural and conformational aspects of the human estrogen receptor has recently begun.