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：
7681461
负责人：
B TRACY NIXON
金额：
$ 31.54万
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 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7681461
关键词：
ATP Hydrolysis ATP phosphohydrolase Address Affect Amino Acid Substitution Arts Bacteria Bacterial RNA Binding Biological Process Biophysics Borrelia burgdorferi Catalysis Cell division Cells Complex Coupling DNA DNA-Directed RNA Polymerase Data Defect Disease Equilibrium Escherichia coli Fluorescence Foundations Funding Future Gene Expression 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 Processed Genes Proteins RNA polymerase sigma 54 Regulation Relative (related person)Research Research Personnel Roentgen Rays Role Sigma Factor Solutions Staging Structure Surface Techniques Testing Time Work Yang analog base information processing insight melting millisecond novel pathogen programs promoter protein complex repaired research study single molecule time interval tool

项目摘要

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.

描述（由申请人提供）：AAA+ ATP酶将ATP水解转化为机械功。很明显，人类细胞和致病病原体都使用这些蛋白质复合物来物理操纵其他蛋白质或 DNA，以拆卸和重新组装膜或其他细胞器、复制 DNA 和遍历细胞分裂、修复受损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。这些知识至关重要，不仅对于我们对一般能量耦合的基本理解，而且对于操纵这些蛋白质以促进人类健康提供线索。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。描述该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。我们建议使用相互补充的新型系综散射和荧光单分子方法来获取平衡状态下和时间相关的溶液相结构知识。为此，我们将使用高度易处理的 NtrC（来自大肠杆菌）和 NtrC1（来自 Aquifex aeolicus）蛋白质作为模型。这些蛋白质与细菌转录因子 sigma-54 相互作用，重塑 RNA 聚合酶以启动转录。在目标 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。与关键氨基酸取代相关的结构动力学缺陷也将使用单分子光谱方法确定。在目标 II 中，将使用 SAXS/WAXS 和小角中子散射 (SANS) 来识别与激活剂/sigma-54 复合物形成相关的核苷酸依赖性构象变化。这将使我们能够定义这些分子机器中核苷酸依赖性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。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Comparative analysis of activator-Esigma54 complexes formed with nucleotide-metal fluoride analogues.

DOI：
10.1093/nar/gkp541
发表时间：
2009-08
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Burrows PC;Joly N;Nixon BT;Buck M
通讯作者：
Buck M

Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein.

AAA 细菌增强子结合蛋白中前传感器 I 插入序列的功能作用。

DOI：
10.1111/j.1365-2958.2009.06744.x
发表时间：
2009
期刊：
Molecular microbiology
影响因子：
3.6
作者：
Burrows,PatriciaC;Schumacher,Jörg;Amartey,Samuel;Ghosh,Tamaswati;Burgis,TimothyA;Zhang,Xiaodong;Nixon,BTracy;Buck,Martin
通讯作者：
Buck,Martin

Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.