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。主要重点涉及三个领域的应用：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有关，2)配体-大分子相互作用的研究，以及3)为支持研究目标而开发和评估蛋白质和其他生物大分子的结构和动态表征的新方法。过去一年的进展摘要如下： 项目1.在过去的一年里，我们使用核磁共振方法确定了噬菌体蛋白HOT的溶液结构，HOT是大肠杆菌DNA聚合酶III的theta亚基的同源序列。这种酶是大肠杆菌的主要复制聚合酶，在其他方面，它是理解决定复制保真度的因素的重要模型。我们的合作者最近的工作表明，表达热蛋白而不是其theta同源物的转基因大肠杆菌细胞在保真度方面表现出细微而有趣的差异，这些差异与与theta/hot相互作用的校对核酸外切酶epsilon的功能改变有关。令人惊讶的是，尽管HOT的二级结构被发现与报道的theta的二级结构非常相似，但折叠拓扑结构完全不同，因此即使是DALI搜索也没有将HOT识别为theta的结构同源，尽管序列同源性很高。圆二向色性分析表明，随着温度的变化，theta和HEAT都能够可逆地展开和再折叠，而HEAT的熔化温度比theta低6摄氏度。 项目2.配体-大分子相互作用的研究继续集中在II型二氢叶酸还原酶，R67 DHFR，这是一种质粒编码的酶，使含有该质粒的细菌对抗叶酸药物产生抗药性。在过去的一年里，我们制备了U-[13C，15N]R67 DHFR，并指定了大部分共振。我们表征了NADP结合的区域和离解常数。出乎意料的是，我们观察到NADP被该酶缓慢地水解为NAD，这一活性可能为该酶的催化机制提供线索。我们还观察到NADP和NADPH的烟酰胺质子之间的配体间Overhauser效应。 项目3.我们已经证明了在含有U-[2H]和U-[13C，15N]氨基酸的混合介质中通过细菌表达来标记蛋白质氨基酸残基的相关性是可能的。这种方法方便了侧链的分配。

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 involves applications in three areas: 1) understanding how the structural and dynamic behavior of DNA polymerases relates to the fidelity of nucleotide incorporation, 2) studies of ligand-macromolecule interactions, and 3) development and evaluation of new methodologies for the structural and dynamic characterization of proteins and other biological macromolecules in support of the research goals. Progress during the past year is summarized below: Project 1. During the past year, we used NMR methods to determine the solution structure of the phage protein, HOT, which is a sequence homolog of the theta subunit of E. coli DNA polymerase III. This enzyme is the main replicative polymerase of E. coli and, among other things, serves as an important model for understanding the factors that determine replicative fidelilty. Recent work by our collaborators has shown that genetically modified E. coli cells which express the HOT protein rather than its theta homolog exhibit subtle and interesting differences in fidelity that are related to altered function of the proofreading exonuclease epsilon that interactions with theta/HOT. Surprisingly, although the secondary structure of HOT was found to be fairly similar to that reported for theta, the folding topology was completely different, so that even a DALI search did not identify HOT as a structural homolog of theta despite a high level of sequence homology. Circular dichroism analysis indicates that both theta and HOT are able to unfold and refold reversibly as a function of temperature, with HOT having a melting temperature that is 6 degrees C below that of theta. Project 2. Studies of ligand-macromolecule interactions have continued to focus on the Type II dihydrdofolate reductase, R67 DHFR, a plasmid encoded enzyme which confers resistance to anti-folate drugs on the bacteria containing the plasmid. During the past year, we prepared U-[13C,15N] R67 DHFR and assigned most of the resonances. We characterized the region and dissociation constant for NADP binding. Unexpectedly, we observed that NADP was slowly hydrolyzed to NAD by the enzyme, an activity which may provide clues into the catalytic mechanism of this enzyme. We also observed inter-ligand Overhauser effects between the nicotinamide protons of NADP and NADPH. Project 3. We have demonstrated that it is possible to introduce a correlation in the labeling of protein amino acid residues by bacterial expression from a medium containing a mixture of U-[2H] and U-[13C,15N] amino acids. This approach facilitates sidechain assignments.