Nmr Studies Of Biomolecular Structure, Function, And Dyn

该项目利用最先进的NMR光谱学来研究NIEHS，结构生物学实验室和NMR研究小组持续感兴趣的问题。目前的主要重点涉及两个领域的应用：1）了解DNA聚合酶的结构和动力学行为如何与核苷酸掺入的保真度相关，以及2）配体-大分子相互作用的研究。第一个项目的最新进展包括：a）阐明了E. coliDNA聚合酶III是大肠杆菌的主要复制聚合酶。大肠杆菌，和B）测定人DNA聚合酶λ的8 kD裂解酶结构域的溶液结构。第一个项目涉及化学位移映射的酰胺和甲基共振位移的变化，同位素标记的ε 186，结果从添加的θ亚基。观察到的ε 186酰胺共振的最显着的位移是本地化的螺旋α 1，β链2和3，以及附近的α-螺旋7开始的区域。pol λ的裂解酶结构域的结构的测定允许与pol β的相应结构域的结构比对。这使我们能够评估以前的建议，参与特定的残留物的催化机制。 配体-大分子相互作用的研究集中在：a）理解R67二氢叶酸还原酶-II型DHFR与其底物和辅因子的相互作用，和B）硼酸盐与胰蛋白酶和其他酶的相互作用的表征。我们能够获得三元R67 DHFR-NADPH-NADP复合物的配体间NOE，证明NADPH H-4质子与NADP H-4和H-5质子的接近性。我们还意外地发现，这种酶能够催化NADP去磷酸化产生NAD（速度相当慢）。这一观察结果提供了对II型DHFR进行的一些早期NMR研究的见解。我们已经扩大了我们的观察硼酸盐-乙醇-胰蛋白酶配合物与额外的NMR和晶体学研究，证明涉及硼酸盐的三元和四元配合物的形成。这种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.