STUDIES OF THE CONFORMATIONAL FLEXIBILITY OF TAQ AND E COLI DNA POLYMERASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 构象转换是几乎所有已知酶功能的基本特征。小角X射线散射（SAXS）是一种灵敏的检测溶液中大分子大小和构象变化的技术，但在生物化学中的应用较少。它特别适用于NMR可及范围之外的分子（>20 kDa）。在这项研究中，小角X射线散射将被用来检查全球构象状态的几种DNA聚合酶作为不同的溶液条件的功能。Taq聚合酶是PCR（聚合酶链反应）中使用的主要聚合酶。目前存在两种相当不同的Taq聚合酶的晶体结构：伸长构象和紧凑形式。由于两种不同的构象显着改变聚合酶的两个酶活性位点之间的距离，分辨率的形式存在于溶液中具有显着的功能意义。在本研究中，作为溶液条件的函数的分子回转半径（Rg）的测量将确定Taq聚合酶是否可以占据溶液中的任一构象，或者它是否主要占据两种构象中的仅一种。Taq和其他3种DNA聚合酶作为热变性和化学变性的函数的扩增也将被检查。来自对这些结构同源的聚合酶进行的解折叠实验的信息令人惊讶地表明，它们的变性状态在大小上非常不同。SAXS测量将直接检验这一假设。除了聚合酶测量，本研究还将测量酶天冬氨酸转氨甲酰酶（ATCase）的Rg变化作为结合配体的函数。多年来，人们已经知道ATCase在其正常功能周期中在两种不同的构象状态（T和R）之间切换。来自几个实验室的最新数据表明，ATCase中的构象开关可能会改变，同时保留与构象开关相关的变构素功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Conformational switching is an essential feature of the function of nearly all known enzymes. Small angle x-ray scattering (SAXS) is a sensitive technique for detecting size and conformation changes of macromolecules in solution, but it is sparingly used in biochemistry. It is particularly useful for molecules outside the range accessible to NMR (>20 kDa). In this study SAXS will be used to examine the global Conformational states for several DNA polymerases as a function of different solution conditions. Taq polymerase is the predominant polymerase used in PCR (the polymerase chain reaction). There currently exist two rather different crystal structures for Taq polymerase: an elongated conformation and a compact form. Since the two different conformations dramatically change the distance between the two enzymatic active sites of the polymerase, the resolution of which forms exist in solution has significant functional implications. Measurements of the molecular radius of gyration (Rg) as a function of solution conditions in this study will determine if Taq polymerase can occupy either conformation in solution, or if it predominantly occupies only one of the two conformations. The expansion of Taq and 3 other DNA polymerases as a function of thermal and chemical denaturation will also be examined. Information from unfolding experiments performed on these structurally homologous polymerases surprisingly suggests that their denatured states are very different in size. SAXS measurements will directly test this hypothesis. In addition to the polymerase measurements, this study will also measure Rg changes for the enzyme aspartate transcarbamylase (ATCase) as a function of bound ligand. ATCase has been known for many years to switch between two different Conformational states (T and R) during its normal functional cycle. Recent data from several labs suggests the Conformational switch in ATCase may be altered while retaining the allostenc functions associated with the conformational switch.