NMR STUDIES OF BIOMOLECULAR STRUCTURE, FUNCTION AND DYNAMICS

我们继续努力修改和改进核磁共振方法，以 共振峰的归属和分子结构的分析 动力学，并将这些方法应用于与以下相关的问题 环境健康。几种新近开发的获得1-甲基-2-甲氧基苯乙酸乙酯的方法 并将二维核磁共振波谱应用于胆汁的分析。 大鼠经α-萘基处理后得到的酸性加合物 引起胆管坏死的异硫氰酸酯(ANIT)。我们的结果 表明胆盐为5-β，胆酸3-α，6-β，7- 含有C22和C23之间的反式CH=CH双键的β三醇。这个 主要加合物为牛磺酸。两个结构的工作仍在继续 问题，都与免疫系统有关。第一个是协作性 与罗伯特·汉舒马赫博士在亲环素酶上的合作， 免疫抑制药物环孢素A的明显靶点 在过去一年中继续进行的合作研究旨在确定 嘌呤核苷酶活性部位的结构 磷酸化酶(PNPase)，它在细胞的分解代谢中起重要作用。 核苷类药物，用于治疗各种疾病，也用于 这些药物的合成。唯一能够研究的核磁共振技术 与如此大的酶的络合物是转移的NOE(TRNOE)，但是 对这些实验的解释是困难的。我们已经利用了一个 本实验的改装为旋转框架。奥豪瑟效应 (TRROE)，以揭示在TRNOE结果中不易识别的伪像。 首先，对于瓷砖，已经进行了大量的理论计算 时间解释了交换动力学。核磁共振方法也被用于 独立确定分子解离的速率常数 酶-抑制剂复合体，这是一个关键参数，需要 转移NOE实验分析。这些研究的结果是 表明与酶结合的抑制剂的构象很多 与根据晶体结构数据预测的结果不同 配基本身。

We have continued our efforts to modify and improve NMR methodologies for the assignment of resonances and the analysis of molecular structure and dynamics, and to apply these approaches to problems related to environmental health. Several recently developed methods for obtaining 1- and 2-dimensional NMR spectra have been applied to the analysis of bile acid adducts obtained subsequent to the treatment of rats with a-naphthyl isothiocyanate (ANIT) which causes bile duct necrosis. Our results indicate that the bile salt is a 5-beta, cholanic acid 3-alpha, 6-beta, 7- beta triol containing a trans CH=CH double bond between C22 and C23. The principal adduct is tauric acid. Work has continued on two structural problems, both related to the immune system. The first is a collaborative effort with Dr. Robert Handschumacher on the enzyme cyclophilin, the apparent target of the immunosuppressive drug cyclosporin A. A second collaborative study continued during the past year is aimed at determining the structure of the active site of the enzyme purine nucleoside phosphorylase (PNPase), which plays an important role in the catabolism of nucleoside drugs used to treat a variety of illnesses, and is also used in the synthesis of these drugs. The only NMR technique capable of studying complexes with enzymes this large is the transferred NOE (TRNOE), but interpretation of these experiments is difficult. We have utilized a modification of this experiment, the rotating-frame. Overhauser effect (TRROE), to reveal artifacts not easily recognized in the TRNOE results. Extensive theoretical calculations have been developed that for tile first time account for exchange kinetics. NMR methods have also been used to independently determine the rate constant for the dissociation of the enzyme-inhibitor complex, which is a critical parameter needed for the analysis of transferred NOE experiments. The results of these studies have shown that the conformation of the inhibitor bound to the enzyme is much different than that predicted on the basis of crystal structure data for the ligands themselves.