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

阐明 AAA ATP 酶 NtrC1 和 NtrC 的构象动力学

基本信息

批准号：
7492548
负责人：
B TRACY NIXON
金额：
$ 1.28万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7492548
关键词：
ATP Hydrolysis ATP phosphohydrolase Address Affect Amino Acid Substitution Arts Bacteria Binding Biological Process Biophysics Borrelia burgdorferi Catalysis Cell division Cells Class Complex Coupling DNA DNA-Directed RNA Polymerase Data Defect Disease Equilibrium Escherichia coli Fluorescence Foundations Funding Future Gene Expression Gene Expression Regulation Genetic Transcription Goals Health Human Human Genome Hydrolysis Kinetics Knowledge Lyme Disease Maps Mechanics Mediating Membrane Methodology Methods Modeling Molecular Biology Molecular Conformation Molecular Machines Mutation Neutrons Nucleotides Organelles Outcome Phase Positioning Attribute Protein Dynamics Proteins RNA RNA polymerase sigma 54 Relative (related person)Research Research Personnel Roentgen Rays Role Sigma Factor Solutions Staging Structure Surface Techniques Testing Time Work Yang analog base insight melting millisecond novel pathogen programs promoter repaired research study single molecule time interval tool

项目摘要

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.

AAA+ AT酶将 ATP 水解转化为机械功。很明显，人体细胞和疾病导致病原体利用这些蛋白质复合物来物理操纵其他蛋白质或 DNA 拆卸并重新组装膜或其他细胞器，以复制 DNA 并遍历细胞分裂，以修复受损的蛋白质，或调节基因表达。这些分子的结构基础然而，机器将 ATP 水解转化为机械功尚不清楚。这些知识并不重要不仅有助于我们对一般能量耦合的基本理解，而且还为我们提供线索操纵这些蛋白质来促进人类健康。事实上，许多疾病都与身体缺陷有关。人类基因组中编码的 80 种 AAA+ ATP 酶中的一种或多种。的一个主要障碍描述机制的原因是我们无法探测相关的详细构象变化 ATP 结合、水解和产物释放的步骤。我们假设这些 ATP 酶的缺陷将这些分子机器在不同阶段循环的方式表现出来 ATP 水解。我们建议使用新颖的整体散射和荧光单分子方法，它们是相互补充的，以获取解决阶段的结构知识平衡并且以时间依赖的方式。为此，我们将使用高度易处理的 NtrC（来自大肠杆菌大肠杆菌）和 NtrC1（来自 Aquifex aeolicus）蛋白质作为模型。这些蛋白质与细菌相互作用转录因子 sigma-54，重塑 RNA 聚合酶以启动转录。在《目标一》中，与不同催化阶段相关的构象变化将使用小和宽来识别角 X 射线散射（SAXS 和 WAXS）。与关键相关的结构动力学缺陷氨基酸取代也将使用单分子光谱方法来确定。在目标二中，与形成相关的核苷酸依赖性构象变化激活剂/sigma-54复合物将使用SAXS/WAXS和小角中子散射进行识别（SANS）。这将使我们能够定义核苷酸依赖性构象变化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。