ATPASE KINETICS AND MACROMOLECULAR ASSEMBLIES OF S54-DEPENDENT, 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。 在以前的项目中，我们建立了两种调控EBP ATPase的机制，定义了它们在催化循环中发生的结构变化，并正在通过对ATPase突变形式的结构功能研究来探讨其潜在的机制。在滴定ADP-BeFx和记录NtrC1ATPase的SAXS谱变化时，观察到了一个有趣但意想不到的结果。观察到了两个不同的构象变化阶段。第一个包括将Rg从47个减少到42个，因为大约一半的可用核苷酸结合位点被占据。第二阶段，仅在第一次转变完成并占据额外的核苷酸结合位点后发生，涉及较高Q值时SAXS谱的变化。

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 prior projects, we established two mechanisms for regulating the EBP ATPases, defined structural changes occuring in their catalytic cycle, and are addressing the underlying mechanism via structure function studies of mutant forms of ATPase. A fascinating yet unexpected outcome was observed upon titrating ADP-BeFx and recording changes in the SAXS profile of the NtrC1 ATPase. Two distinct phases of conformational change were observed. The first involved reducing Rg from 47 ¿ to 42 ¿ as about half of the available nucleotide binding sites became occupied. The second phase, only occurring after the first transition was completed and additional nucleotide binding sites became occupied, involved change in the SAXS profile at higher Q values.