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 去年以来，该项目的重点主要集中在两个方面：(1) 转移NOE(TRNOE)技术的发展及其在石油化工中的应用 核苷类药物杀菌素的构象测定 与大肠杆菌嘌呤核苷磷酸化酶(PNPase)的复合体，和(2) 苯硼酸(BBA)衍生物与苯乙醇胺的相互作用分析 小分子和带有枯草杆菌酶的。的一个重要目标 这两个问题都是动力学行为的特征。 具体地说，我们之前已经确定了TRNOE参数 与大分子结合部位交换的抑制剂可以是 对汇率的依赖性很强。我们最近评估了 使用两种策略来确定配基交换率：(1) 使用CPMG技术作为脉率的函数来测量T2，以及 (2)作为配基的函数的配体的T1O值的测量 自旋锁定场的强度。我们目前正在进行详细的 PNPase中这些实验的理论和实验分析 杀瘤蛋白系统。在第二个领域，对BBA衍生品的研究 用不同的生物学方法测定表观离解常数 生理条件下的重要配体以及结合 运动学。4-氟-BBA与丝氨酸蛋白酶的结合动力学 枯草杆菌菌素与在不同模型配体上得到的结果进行了比较 以表征与酶的相互作用。

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 primary emphasis of this project has been in two areas: (1) 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), and (2) analysis of the interaction of benzeneboronic acid (BBA) derivatives with small molecules and with the enzyme subtilisin. An important objective of both problems was a characterization of the kinetic behavior. Specifically, we have previously determined that the TRNOE parameters for inhibitors which are exchanging with macromolecular binding sites can be strongly dependent on the exchange rates. We have recently evaluated the use of two strategies for determining ligand exchange rates: (1) Measurement of T2 using the CPMG technique as a function of pulse rate, and (2) measurement of the T1o values for the ligand as a function of the strength of the spin-lock field. We are currently carrying out detailed theoretical and experimental analyses of these experiments in the PNPase - tubercidin system. In the second area, studies with BBA derivatives have determined apparent dissociation constants with various biologically significant ligands under physiological conditions, as well as binding kinetics. The binding kinetics of 4-fluoroBBA with the serine protease subtilisin were compared with results obtained on various model ligands in order to characterize the interaction with the enzyme.