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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 构象转换是几乎所有已知的酶功能的基本特征。小角X射线散射(SAXS)是一种检测溶液中大分子尺寸和构象变化的灵敏技术，但在生物化学中的应用较少。它对核磁共振(&gt；20 kDa)可及范围以外的分子特别有用。在这项研究中，SAXS将被用来检测几种DNA聚合酶的整体构象状态作为不同溶液条件的函数。Taq聚合酶是聚合酶链式反应中最主要的聚合酶。目前，Taq聚合酶有两种截然不同的晶体结构：拉长的构象和紧凑的构象。由于两种不同的构象极大地改变了聚合酶两个酶活性部位之间的距离，因此溶液中存在的形式的分辨具有重要的功能意义。在这项研究中，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何一种构象，或者它是否主要占据两种构象中的一种。此外，还将研究Taq和其他3种DNA聚合酶在热变性和化学变性过程中的膨胀情况。对这些结构上同源聚合酶进行的展开实验所获得的信息令人惊讶地表明，它们的变性状态在大小上有很大不同。SAXS的测量将直接验证这一假设。除了聚合酶测量，这项研究还将测量天冬氨酸氨基转移酶(ATCase)作为结合配体的功能的Rg变化。多年来，人们已经知道ATCase在其正常的功能周期中会在两种不同的构象状态(T和R)之间切换。几个实验室的最新数据表明，ATCase中的构象开关可能会发生改变，同时保留与构象开关相关的同种异体功能。

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.