Aids Related NMR Research

该项目利用核磁共振光谱学研究艾滋病毒和模型系统的分子组成部分。主要研究方向为：1）HIV逆转录酶核糖核酸酶H结构域的结构、动力学和配体结合行为分析; 2）模型核酸酶和聚合酶系统的研究。在过去的一年中，我们克隆和表达了同位素标记的HIV-2逆转录酶的U-[13 C，15 N] RNase H结构域。可以预期，来自HIV-1和HIV-2的RNA酶H结构域的详细结构和动力学比较可以提供对该结构域的溶液行为的洞察，其可用于药物开发。我们还研究了该结构域与疏水分子的相互作用，以确定潜在的配体结合位点。作为上述第二个目标的一部分，我们一直对模型核酸酶抑制剂系统的研究感兴趣，作为理解核酸酶抑制所涉及的原理的基础。我们先前已经确定了来自鱼腥藻的核酸酶A（NucA）抑制剂NuiA的溶液结构。在过去的一年中，我们已经确定了结构的NucA的X射线晶体学。虽然最初计划作为NMR研究，但筛选改善溶解度的溶液条件导致该酶意外结晶，这被用来获得晶体结构。NucA的结构，这表明相当大的结构同源性与沙雷氏菌核酸酶来自粘质沙雷氏菌，而共享只有22%的序列同一性，然而，是一个单体，而不是一个二聚体，也揭示了一个意想不到的额外的二价金属离子的结合位点位于~ 26？从活性酶的位置。酶结构的可用性提供了对催化机制的深入了解，目前正在进行研究以获得NucA-NuiA核酸酶-抑制剂复合物的结构。除了这项工作，我们有一个持续的兴趣在DNA修复酶波尔？作为HIV逆转录酶的模型系统。我们以前已经证明，NMR研究[甲基-13 C]蛋氨酸标记波尔？为研究酶对核苷酸底物的反应提供了有用的探针。在过去的一年中，我们研究了酶的DNA类似物，其中胸苷电子等排体-二氟甲苯-被引入到模板碱基位置的响应。在加入dATP以形成失败的三元复合物时，酶不能显示正常的构象激活，如果胸腺嘧啶在模板碱基位置，则会观察到正常的构象激活。该观察结果解释了在这些条件下酶不能进行聚合反应。

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. The primary research areas are: 1) analysis of the structure, dynamics and ligand binding behavior of the Ribonuclease H domain of HIV reverse transcriptase, and 2) studies of model nuclease and poymerase systems. During the past year, we have cloned and experessed the isotopically labeled, U-[13C,15N] RNase H domain of reverse transcriptase derived from HIV-2. It is anticipated that a detailed structural and dynamic comparison of the RNase H domains derived from HIV-1 and HIV-2 can provide insight into the solution behavior of this domain which can be used for drug development. We have also studied the interaction of this domain with hydrophobic molecules in order to identify potential ligand binding sites. As part of the second objective noted above, we have been interested in studies of model nuclease-inhibitor systems as a basis for understanding the principles involved in nuclease inhibition. We had previously determined the solution structure of NuiA, an inhibitor of Nuclease A (NucA) from Anabaena. During the past year, we have determined the structure of NucA by X-ray crystallography. Although initially planned as an NMR study, the screening of solution conditions for improved solubility led to the unexpected crystallization of this enzyme, which was exploited to obtain the crystal structure. The structure of NucA, which shows considerable structural homology with the Serratia nuclease derived from Serratia marcescens while sharing only 22% sequence identity, nevertheless is a monomer rather than a dimer, and also revealed an unanticipated additional binding site for divalent metal ions located ~ 26 ? from the active enzyme site. The availability of the enzyme structure provides insight into the catalytic mechansim, and studies are currently in progress to obtain the structure of the NucA-NuiA, nuclease-inhibitor complex. In addition to this work, we have a continuing interest in the DNA repair enzyme Pol ? as a model system for HIV reverse transcriptase. We have previously demonstrated that NMR studies of [methyl-13C]methionine labeled Pol ? provides a useful probe for studying the response of the enzyme to nucleotide substrates. During the past year, we studied the response of the enzyme to a DNA analog in which a thymidine isostere - difluorotoluene - was introduced into the templating base position. Upon addition of dATP to form an abortive ternary complex, the enzyme fails to show the normal conformational activation that would be observed if thymine were in the templating base position. This observation explains the failure of the enzyme to proceed with the polymerization reaction under these conditions.