ATPASE KINETICS AND MACROMOLECULAR ASSEMBLIES OF S54-DEPENDENT, AAA+ ATPASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 为什么分子机器（AAA+ ATP酶）的缺陷会导致疾病？这些蛋白质机器将ATP水解转化为机械功。人类细胞和致病病原体都利用这项工作来物理操纵蛋白质或DNA，以拆除和重新组装膜或其他细胞器，复制DNA并穿越细胞分裂，修复受损蛋白质或调节基因表达。我们不知道这些分子机器如何将ATP水解转化为机械功。我们的研究集中在AAA+ ATPAes的一个子集，细菌增强子结合蛋白（EBP），利用其ATP酶活性调节有害活动（疾病，作物损害）或有益活动（固氮，环境修复，氢或其他代谢产物生产）所需的基因转录。 在先前的项目中，我们建立了两种调节EBP ATP酶的机制，定义了其催化循环中发生的结构变化，并通过ATP酶突变形式的结构功能研究来解决潜在的机制。在滴定ADP-BeFx并记录NtrC 1 ATP酶SAXS谱的变化时，观察到了一个有趣但意想不到的结果。观察到两个不同的构象变化阶段。第一个是将Rg从47减少到42，因为大约一半的可用核苷酸结合位点被占据。第二阶段，只发生在第一个过渡完成后，额外的核苷酸结合位点被占用，涉及在较高的Q值的SAXS配置文件的变化。

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.