Aids Related NMR Research

这个项目利用核磁共振光谱学来研究HIV的分子组成。我们制备了[甲基-13C]蛋氨酸标记的病毒逆转录酶P51亚基，并用核磁共振波谱对该体系进行了研究。我们能够观察到这个亚基中六个蛋氨酸残基中的五个，目前正在进行研究，以确定这些共振。特别感兴趣的是活性P66亚基和非活性P51亚基之间的YMDD基序的结构比较。我们还开始了对逆转录酶的RNaseH结构域的研究。先前的核磁共振研究表明，分离的结构域在其结构中表现出广泛的迁移率。我们目前正在研究镁离子对隔离结构域内部迁移率的影响。除了对HIV逆转录酶及其结构域的研究外，我们还利用核磁共振波谱对II型二氢叶酸还原酶进行了研究。免疫系统受损的患者更容易受到外来细菌感染，导致对抗生素治疗的依赖增加。细菌对抗叶酸药物治疗耐药的一种机制涉及表达一种质粒II型二氢叶酸还原酶，该酶在结构上与染色体DHFR无关，并对针对染色体酶的细菌抗叶酸如甲氧普林具有耐药性。我们一直在研究II型DHFR与配体的相互作用，以了解其催化机理，并开发新的抑制剂。观察到连接吡啶核苷酸和叶酸质子的配体间Overhaser效应，并用于确定DHFR-NADP-叶酸三元络合物的结构。我们还制备了[U-13C，15N]-DHFR，并正在研究标记酶与底物和辅因子的相互作用。

This project utilizes NMR spectroscopy to study the molecular components of HIV. We have prepared [methyl-13C]methionine labeled P51 subunit of the viral reverse transcriptase, and studied this system by NMR spectroscopy. We were able to observe five of the six methionine residues in this subunit, and are currently performing studies to assign these resonances. Of particular interest is a comparison of the structure of the YMDD motif between the active, P66 subunit and the inactive P51 subunit. We also have begun studies on the RNase H domain of the reverse transcriptase. Previous NMR studies have demonstrated that the isolated domain exhibits extensive mobility throughout its structure. We are currently studying the effects of magnesium ion on the internal mobility of the isolated domain. In addition to the studies of HIV reverse transcriptase and its domains, we have been using NMR spectroscopy to study Type II Dihydrofolate Reductase. Patients with a compromised immune system are more susceptible to adventitious bacterial infections, leading to increased dependence on antibiotic treatments. One mechanism of bacterial resistance to anti-folate drug therapy involves the expression of a plasmid type II dihydrofolate reductase, which is structurally unrelated to chromosomal DHFR and is resistant to bacterial antifolates such as trimethoprim, which target the chromosomal enzyme. We have been studying the interaction of the type II DHFR with ligands in order to understand the catalytic mechanism and to develop new inhibitors. Inter-ligand Overhaser effects connecting the pyridine nucleotide and folate protons were observed and have been used to define the structure of the ternary DHFR-NADP-folate complex. We also have prepared [U-13C,15N]-DHFR and are studying the interaction of the labeled enzyme with substrate and cofactor.