SOLUTION STRUCTURE OF CHLORELLA VIRUS DEOXYURIDINE TRIPHOSPHATE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这种酶显示了一个典型的水平基因转移的例子，它模仿了癌症的一个发展阶段。DUTPase活性的缺乏启动了胸腺嘧啶脱氢酶细胞的死亡。对该酶的机制和细胞作用的结构洞察在调节无胸腺嘧啶的细胞凋亡中至关重要。通过结合溶液X射线散射和结晶学研究，我们已经开始研究影响小球藻病毒dUTPase最适温度的分子机制，每个dUTPase都表现出最佳催化活性的不同温度。森山团队最近解决了该酶的晶体结构和一种突变酶的最适温度与野生型酶不同的问题。我们最近进行了初步的溶液X射线散射实验，获得了野生型的35.5°和突变体的39.1°的回转半径。这些值大于晶体结构的期望值，可能是由于组氨酸标签的存在有助于纯化。然而，这些蛋白质在全球结构上的实质性差异与我们的结晶学研究提出的构象差异是一致的。我们计划将溶液散射研究扩展到更高的结构分辨率，并研究溶液散射与温度依赖性酶活性的关系。

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. This enzyme shows a typical example of horizontal gene transfer, which mimics a developmental stage of cancers. The lack of dUTPase activity initiates thymine-less cell death. Structural insights on the mechanism and cellular role of the enzyme are crucial in modulating thymine-less apoptosis. We have begun studying the molecular mechanisms which influence optimum temperatures of two dUTPases from the chlorella virus, each exhibiting different temperature for optimum catalytic activity, by combining solution structure deduced by solution x-ray scattering and crystallographic studies. The Moriyama group has recently solved the crystal structure of the enzyme and a mutant enzyme whose optimum temperature differs from that of the wild type enzyme. We recently conducted preliminary solution x-ray scattering experiments and obtained radii of gyration 35.5 ¿¿for the wild type and 39.1¿¿for the mutant. These values are larger than what can be expected from the crystallographic structures possibly due to the presence of a histidine tag to aid purification. However, the substantial difference in global structures for these proteins is consistent with the conformational difference being suggested by our crystallographic studies. We are planning on extending the solution scattering study to higher structural resolution, and examine how solution scattering correlates with the temperature dependent enzyme activity.