STRUCTURE - FUNCTION AND KINETICS OF AAA+ ATPASES

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。 为什么分子机器(AAA+ATPase)中的缺陷会导致疾病？这些蛋白质机器将三磷酸腺苷的水解转化为机械功。人类细胞和引起疾病的病原体都利用这种工作来物理地操纵蛋白质或DNA，以拆解和重组膜或其他细胞器，复制DNA并穿越细胞分裂，修复受损的蛋白质，或调节基因表达。我们不知道这些分子机器是如何将三磷酸腺苷的水解转化为机械功的。我们的研究集中在AAA+ATPase的一个子集，细菌增强子结合蛋白(EBPs)，它利用它们的ATPase活性来调节有害活动(疾病、作物损害)或有用活动(固氮、环境修复、氢气或其他代谢物产生)所需基因的转录。 在之前的一个项目中，我们建立了两种调节EBP ATPase的机制，并开始定义在其催化循环中发生的结构变化。目前的项目完成了后一个目标，并将其扩展到通过对ATPase突变形式的结构功能研究来解决潜在的机制。收集了突变蛋白质的初始数据，最终数据收集计划在2007年3月进行。此外，2006年收集了一种新的ATPase与其靶蛋白(RNA聚合酶的s54亚基)的复合体的数据，并对启动子DNA与s54、s54与ATPase的复合体或s54、ATPase与RNA聚合酶的其余部分进行了对比变异观察。最后，获得了另外两个EBP蛋白和一个真核细胞染色质重塑蛋白的结构的初步数据。

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. Why do defects in molecular machines (AAA+ ATPases) cause disease? These protein machines convert ATP hydrolysis into mechanical work. Both human cells and disease causing pathogens use this work to physically manipulate proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. We do not know how these molecular machines convert ATP hydrolysis into mechanical work. Our research focuses on one subset of AAA+ ATPAses, the bacterial-enhancer-binding proteins (EBPs) which use their ATPase activities to regulate transcription of genes needed for harmful activites (diseases, crop damage) or helpful ones (nitrogen fixation, environmental remediation, hydrogen or other metabolite production). In a prior project, we established two mechanisms for regulating the EBP ATPases, and began defining structural changes occuring in their catalytic cycle. The current project completes this latter goal and extends it to address the underlying mechanism via structure function studies of mutant forms of ATPase. Initial data for mutant proteins were collected, with final data collection scheduled for March of 2007. In addition, in 2006 data were collected for a novel complex of ATPase and its target protein (the s54 subunit of RNA polymerase), and control experiments for contrast variation SANS observations of complexes of promoter DNA with s54, s54 and ATPase, or s54, ATPase and the rest of RNA polymerase. Finally, preliminary data for structures of two additional EBP proteins and a eukaryotic chromatin remodeling protein were obtained.