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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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，但在生物化学中应用较少。它特别适用于核磁共振（bbb20 kDa）范围以外的分子。在本研究中，SAXS将用于检查几种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PCR（聚合酶链反应）中使用的主要聚合酶。目前Taq聚合酶存在两种不同的晶体结构：细长构象和致密构象。由于两种不同的构象极大地改变了聚合酶的两个酶活性位点之间的距离，因此在溶液中存在哪种构象的解决具有重要的功能意义。在本研究中，分子旋转半径（Rg）作为溶液条件的函数的测量将确定Taq聚合酶是否可以在溶液中占据任何一种构象，或者它是否主要占据两种构象中的一种。Taq和其他3个DNA聚合酶的扩展作为热变性和化学变性的功能也将被检查。对这些结构同源聚合酶进行的展开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。除了聚合酶的测量外，本研究还将测量天冬氨酸转甲氨基酰基酶（ATCase）的Rg变化作为结合配体的函数。多年来，人们已经知道，在正常的功能周期中，ATCase可以在两种不同的构象状态（T和R）之间切换。最近来自几个实验室的数据表明，ATCase的构象开关可能被改变，同时保留与构象开关相关的异速功能。

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.