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

• 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
• 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

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+ATPase将ATP的水解功转化为机械功。很明显，人类细胞和疾病 导致病原体使用这些蛋白质复合体来物理操作其他蛋白质或DNA来 拆卸和重组细胞膜或其他细胞器，以复制DNA并穿越细胞分裂，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为人所知。这样的知识不是至关重要的 不仅有助于我们对能量耦合的基本理解，还有助于为 操控这些蛋白质以促进人类健康。事实上，许多疾病都与基因缺陷有关。 编码在人类基因组中的80个AAA+ATPase中的一个或多个。一个主要的障碍 描述这种机制的原因是我们无法探索与之相关的详细构象变化 到三磷酸腺苷结合、水解和产物释放的步骤。我们假设这些ATPase中的缺陷将 以这些分子机器在不同阶段循环的方式表现出来 三磷酸腺苷水解。我们建议使用新的系综散射和荧光单分子方法， 它们是相辅相成的，以获取解决方案阶段结构知识，两者都在 均衡，并以时间依赖的方式。为此，我们将使用高度易处理的NTRC(来自Escherichia Coli)和Ntrc1(Aquifex Aeolicus)蛋白作为模型。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AIM I中， 与催化的不同阶段相关的构象变化将通过使用小的和广泛的- 角X射线散射(SAXS和WAXS)。与关键因素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AIM II中， 与核糖核酸相关的构象变化 激活剂/Sigma-54复合体将使用SAXS/WAXS和小角中子散射进行鉴定 (SANS)。这将使我们能够定义核苷酸依赖的构象变化在 这些分子机器。在进行这项研究的过程中，将开发新的工具，这些工具 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。