Elucidating the Conformational Dynamics of the AAA+ ATPases NtrC1 and NtrC

AAA+ ATP酶将ATP水解转化为机械功。很明显，人类细胞和疾病 导致病原体使用这些蛋白质复合物物理操纵其他蛋白质或DNA， 拆卸和重组膜或其他细胞器，复制DNA和穿越细胞分裂， 修复受损的蛋白质或调节基因表达。这些分子的结构基础 然而，机器将ATP水解转化为机械功是未知的。这些知识至关重要， 这不仅有助于我们对能量耦合的基本理解，而且也为我们提供了线索， 操纵这些蛋白质来促进人类健康。事实上，许多疾病都与基因缺陷有关。 人类基因组中编码的80种AAA+ ATP酶中的一种或多种。的主要障碍 描述机制一直是我们无法探测详细的构象变化， 到ATP结合、水解和产物释放的步骤。我们假设这些ATP酶的缺陷会 以这些分子机器循环通过不同阶段的方式表现出来， ATP水解。我们建议使用新的系综散射和荧光单分子方法， 这两种方法是相辅相成的，在两种情况下都可以获得溶液相结构知识。 平衡和时间依赖的方式。为此，我们将使用高度易处理的NtrC（来自埃希氏菌（Escherichia coli）和NtrC 1（来自Aquifex aeolicus）蛋白作为模型。这些蛋白质与细菌 转录因子sigma-54可以重塑RNA聚合酶以启动转录。在Aim I中， 与不同阶段的催化作用有关的构象变化将被识别， 角X射线散射（SAXS & WAXS）。结构动力学中的缺陷， 氨基酸取代也将使用单分子光谱方法来确定。在Aim II中， 与这些蛋白质的形成相关的核苷酸依赖性构象变化 活化剂/σ-54复合物将使用SAXS/WAXS和小角中子散射来鉴定 （SANS）。这将使我们能够确定核苷酸依赖性构象变化在细胞内的功能作用。 这些分子机器在进行这项研究的过程中，将开发新的工具， 预计将广泛适用于对人类健康至关重要的其他蛋白质的类似研究。

AAA+ ATPases convert ATP hydrolysis into mechanical work. It is clear that both human cells and disease causing pathogens use these protein complexesto physically manipulate orther 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. The structural basis on which these molecular machines convert ATP hydrolysis into mechanical work, however, is not known. Such knowledge is vital not only to our fundamental understanding of energy coupling in general, but also to providing clues to manipulate these proteins to promote human health. Indeed, many diseases are associated with defects in one or more of the 80 AAA+ ATPases that are encoded in the human genome. A major impediment to delineating the mechanisms has been our inability to probe detailed conformational changes that are related to steps in ATP binding, hydrolysis, and product release. We hypothesize that defects in these ATPases will manifest themselves in the manner by which these molecular machines cycle through different stages of ATP hydrolysis. We propose to use novel ensemble scattering and fluorescence single-molecule methods, which are complementary to each other, to aquire solution-phase structural knowledge both under equilibrium and in a time-dependent way. To this end, we will use the highly tractable NtrC (from Escherichia coli) and NtrC1 (from Aquifex aeolicus) proteins as models. These proteins interact with the bacterial transcriptional factor, sigma-54, to remodel RNA polymeraseto initiate transcription. In Aim I, the conformational changes associatedwith different stages of catalysis will be identified using small- and wide- angle x-ray scattering (SAXS & WAXS). Defects in structural dynamics that are associated with crucial amino acid substitutions will also be determined using single-molecule spectroscopic approaches. In Aim II, the nucleotide-dependent conformational changes that are associated with the formation of the activator/sigma-54 complex will be identified using both SAXS/WAXS and small-angle neutron scattering (SANS). This will allow us to define the functional roles of nucleotide-dependent conformational changes in these molecular machines. In the course of performing this research, new tools will be developed that are expected to be broadly applicable to similar studies of other proteins that are vital for human health.