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

描述（由申请人提供）：AAA+ ATP酶将ATP水解转化为机械功。很明显，人类细胞和引起疾病的病原体都使用这些蛋白质复合物来物理地操纵其他蛋白质或DNA来拆除和重新组装膜或其他细胞器，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。然而，这些分子机器将ATP水解转化为机械功的结构基础尚不清楚。这些知识是至关重要的，不仅是我们对能量耦合的基本理解，而且还提供了操纵这些蛋白质促进人类健康的线索。事实上，许多疾病都与人类基因组中编码的80种AAA+ atp酶中的一种或多种缺陷有关。描述机制的一个主要障碍是我们无法探测与ATP结合、水解和产物释放步骤相关的详细构象变化。我们假设这些ATP酶的缺陷将以这些分子机器在ATP水解的不同阶段循环的方式表现出来。我们建议使用新的集合散射和荧光单分子方法，这是互补的，以获得平衡和时间相关的溶液相结构知识。为此，我们将使用高度可处理的NtrC（来自大肠杆菌）和NtrC1（来自水蛭）蛋白作为模型。这些蛋白与细菌转录因子sigma-54相互作用，重塑RNA聚合酶以启动转录。在Aim I中，将使用小角和广角x射线散射（SAXS和WAXS）来识别与不同催化阶段相关的构象变化。结构动力学中与关键氨基酸取代相关的缺陷也将使用单分子光谱方法确定。在Aim II中，将使用SAXS/WAXS和小角中子散射（SANS）来识别与活化剂/sigma-54复合物形成相关的核苷酸依赖性构象变化。这将使我们能够确定这些分子机器中依赖核苷酸的构象变化的功能作用。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。

DESCRIPTION (provided by applicant): AAA+ ATPases convert ATP hydrolysis into mechanical work. It is clear that both human cells and disease causing pathogens use these protein complexes to 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 polymerase to initiate transcription. In Aim I, the conformational changes associated with 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.