STRUCTURE AND DYNAMICS OF ASIA AND THE ASIA 670 COMPLEX

亚洲和亚洲 670 综合体的结构和动态

• 批准号: 6797971
• 负责人: JEFFREY L URBAUER
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6797971
• 关键词: DNA directed RNA polymerase; bacterial proteins; bacteriophage T7; binding sites; genetic transcription; nuclear magnetic resonance spectroscopy; physical model; protein protein interaction; protein structure function; structural biology; thermodynamics; virus protein

The antibiotic consequences of the presence of the AsiA protein product of the asiA gene bacgteriophage T4, are due to its tight interaction with bacteral sigma factors, an example of which is its intaration with sigma 70 of E. Coli. The overall goal of the proposed research is to provide a structural and dynamic platform for understanding, at atamoic resolution, the interation between AsiA and bacterial sigma factors, and as result, to elucidate the mechanism of regulation of bacterial transcription by AsiA. Besides being intrinically relevant to furthering our understanding of the fundamentsls of portein-protein interactions in general, the results are an obligatory prelude to the design of a novel broad spectrumantibioic exploiting AsiA mimicry. The binding of the AsiA protein to the sigma 70 subunit of the E. Coli RNA polymerase is clearly one of the principle factors governing the direction and progression of transcription of the T4 genome. In addition, because of lethal effects on bacteria of overexpression of AsiA, and AsiA-sigma 70 interface could represent the prototype for an exciting novel class of borad spectrum antibiotics and it's target. The research proposed will examine the structure and dynamics of AsiA in solution alone and ocmplexed to the AsiA binding domain of the sigma 70 subunit of the e. Coli RNA polymerase, using primarily nuclear magnetic resonance (NMR) techniques. High resolution structural models fo rthe AsiA protein and its complex with the AsiA binding domain of sigma 70 will be generated using distance geometry/simulated annealing methods and NMR-derieved restraints. Hydrogen exchange and 15N and 13C relatxation techniques will be used to determine the distribution, magnitude, and timescale of motions of the backbone and side chains of AsiA and the AsiA sigma 70 complex. Binding studies and novel high pressure NMR studies wil reveal the site-specific thermodynamics of the AsiA-sigma70 interaction, will provide an intricate account of the static and dynamic events interactions in gerneral, and will provide the critical chemical view necessary for pursuit of structure-based drug design to mimic the antibiotic properties of AsiA.

asiA 基因噬菌体 T4 的 AsiA 蛋白产物存在的抗生素后果是由于其与细菌 sigma 因子的紧密相互作用，其中一个例子是它与大肠杆菌的 sigma 70 的结合。 本研究的总体目标是提供一个结构和动态平台，用于在原子分辨率下理解AsiA和细菌西格玛因子之间的相互作用，从而阐明AsiA调节细菌转录的机制。 除了本质上与进一步加深我们对蛋白质-蛋白质相互作用的基础知识的理解相关之外，这些结果是设计一种利用 AsiA 拟态的新型广谱抗生素的必然前奏。 AsiA 蛋白与大肠杆菌 RNA 聚合酶的 sigma 70 亚基的结合显然是控制 T4 基因组转录方向和进展的主要因素之一。 此外，由于AsiA的过度表达对细菌具有致命作用，AsiA-sigma 70界面可以代表一类令人兴奋的新型硼谱抗生素及其靶点的原型。 拟议的研究将检查单独溶液中的 AsiA 以及与 e 的 sigma 70 亚基的 AsiA 结合域复合的结构和动力学。大肠杆菌 RNA 聚合酶，主要使用核磁共振 (NMR) 技术。 使用距离几何/模拟退火方法和 NMR 衍生的限制，将生成 AsiA 蛋白及其与 sigma 70 的 AsiA 结合域的复合物的高分辨率结构模型。 氢交换以及 15N 和 13C 松弛技术将用于确定 AsiA 和 AsiA sigma 70 复合体的主链和侧链运动的分布、幅度和时间尺度。 结合研究和新颖的高压核磁共振研究将揭示AsiA-sigma70相互作用的位点特异性热力学，将提供一般静态和动态事件相互作用的复杂解释，并将提供追求基于结构的药物设计以模拟AsiA抗生素特性所需的关键化学观点。