Nmr Studies Of Biomolecular Structure, Function, And Dyn

该项目利用最先进的核磁共振波谱来研究NIEHS、结构生物学实验室和核磁共振研究小组持续感兴趣的问题。目前的重点主要涉及两个领域的应用：1)了解DNA聚合酶的结构和动态行为如何与核苷酸掺入的保真度相关；2)研究配体-大分子相互作用。第一个项目的最新进展包括：a)阐明了大肠杆菌DNA聚合酶III的epsilon和theta亚基的结合界面，以及b)确定了人DNA聚合酶lambda的8 kD裂解酶结构域的溶液结构。第一个项目涉及酰胺的化学位移图谱和同位素标记的epsilon186中的甲基共振位移变化，这些变化是由theta亚单位的添加引起的。观察到的最显著的epsilon186酰胺共振位移位于螺旋1，β链2和3，以及靠近α-螺旋7开始的区域。Pollambda裂解酶结构域的结构的确定使其与olβ的相应结构域保持了结构上的一致性。这使我们能够评估以前关于特定残基参与催化机制的建议。 配基-大分子相互作用的研究集中在：a)了解R67二氢叶酸还原酶-a型DHFR与其底物和辅因子的相互作用，以及b)表征硼酸盐与胰蛋白酶和其他酶的相互作用。我们获得了R67 DHFR-NADPH-NADP三元络合物的配体间NOs，证明了NADPH H-4质子与NADP H-4和H-5质子的接近。我们还意外地发现，该酶能够催化NADP的去磷酸化以产生NAD(以相当慢的速度)。这一观察结果为早期对II型DHFR进行的一些核磁共振研究提供了洞察力。我们用额外的核磁共振和结晶学研究扩展了我们对硼酸盐-醇-胰酶络合物的观察，证明了涉及硼酸盐的三元和四元络合物的形成。该方法为酶抑制剂的开发提供了潜在的基础。

This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the NIEHS, the Laboratory of Structural Biology, and the NMR research group. The primary emphasis currently involves applications in two areas: 1) Understanding how the structural and dynamic behavior of DNA polymerases relates to the fidelity of nucleotide incorporation, and 2) studies of ligand-macromolecule interactions. Recent progress on the first project has included: a) Elucidation of the binding interface for the epsilon and theta subunits of E. coli DNA polymerase III - the main replicative polymerase of E. coli, and b) Determination of the solution structure of the 8 kD lyase domain of human DNA polymerase lambda. The first project involved chemical shift mapping of the amide and methyl resonance shift changes in isotopically labeled epsilon186 that result from the addition of the theta subunit. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1, beta strands 2 and 3, and to the region near the beginning of alpha-helix 7. The determination of the structure of the lyase domain of pol lambda has allowed a structural alignment with the corresponding domain of pol beta. This has allowed us to evaluate previous proposals regarding the involvement of specific residues in the catalytic mechanism. Studies of ligand-macromolecule interactions have focused on: a) understanding the interaction of R67 dihydrofolate reductase - a Type II DHFR, with its substrate and cofactor, and b) characterization of the interaction of borate with trypsin and other enzymes. We were able to obtain interligand NOEs for the ternary R67 DHFR-NADPH-NADP complex that demonstrate the proximity of the NADPH H-4 protons to the NADP H-4 and H-5 protons. We also made the unanticipated discovery that the enzyme is able to catalyze the dephosphorylation of NADP to yield NAD (at a fairly slow rate). This observation provides insight into some of the earlier NMR studies that had been performed on Type II DHFR. We have extended our observations of borate-alcohol-trypsin complexes with additional NMR and crystallographic studies that demonstrate the formation of ternary and quaternary complexes involving borate. This approach provides a potential basis for the development of enzyme inhibitors.