Structural and Dynamic Studies of Catabolite Activator Protein Complexes

分解代谢物激活蛋白复合物的结构和动力学研究

• 批准号: 1121896
• 负责人: Charalampos Kalodimos
• 金额: $ 62.22万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1121896&HistoricalAwards=false
• 关键词: Structural; Dynamic; Studies; Catabolite; Activator

Intellectual meritGenetic regulatory proteins target specific sites within the genome and either enhance or repress transcriptional activity to elicit cellular responses. The Escherichia coli catabolite activator protein (CAP; referred to also as the cAMP receptor protein, CRP) is a universal transcriptional activator that regulates the expression of over two hundred genes. CAP has long served as the textbook example for understanding transcription regulation. CAP has provided a classic model system for structural and mechanistic studies of transcription activation. Mechanistic descriptions of transcription activation, developed for CAP, are more nearly complete than descriptions of any other examples of transcription activation. Nevertheless, the complete structural basis for CAP-mediated transcription activation remains unknown. Notably, over the recent years CAP has provided an excellent system in which to examine the structure- and dynamics-function relationships that form the basis of allostery. CAP has provided the first experimentally identified system wherein allosteric interactions are mediated through changes in protein motions, in the absence of changes in the mean structure of the protein. The main objectives of this project are to use CAP as a model system to address fundamental questions regarding allosteric regulation and transcriptional activation. An integrated structural, dynamic, and thermodynamic approach will be used to (1) characterize the dynamics of CAP mutants with altered allosteric properties and their interaction energetics with DNA; (2) determine the solution structure of the class I and class II CAP-dependent promoter subassemblies and (3) determine the structural basis for the assembly of the entire CAP-mediated transcription initiation complex.Broader impactIn addition to addressing fundamental biological questions, this project will be used to train students in structural biology, biophysics, and molecular biology, areas that are rapidly becoming integrated in 21st century science. Postdocs, graduate and undergraduate students will have the opportunity to be involved in a multi-disciplinary project that aims at the development of groundbreaking methodologies to enable the structural and dynamic characterization of supramolecular protein complexes by high resolution NMR spectroscopy. This will enable researchers to approach problems from a multidisciplinary and interactive perspective, thus experiencing first hand the utility of applying state-of-the-art methodologies to important biological problems. The paradigm of combining structural, dynamic, thermodynamic and kinetic approaches to study complex protein systems will be included in a new course, currently designed by the PI, to exemplify the value of using an interdisciplinary and quantitative approach to answer questions of scientific importance. The course is intended for a large, diverse audience consisting of graduate and advanced undergraduate students in the programs of Molecular Biosciences, Chemistry and Chemical Biology, Biomedical Engineering and BIOMAPS at Rutgers University.

智力优势基因调节蛋白针对基因组中的特定位置，增强或抑制转录活性，以引发细胞反应。大肠杆菌分解代谢激活蛋白(CAP；也被称为cAMP受体蛋白，CRP)是一种通用的转录激活蛋白，调节200多个基因的表达。长期以来，CAP一直是理解转录调控的教科书范例。CAP为转录激活的结构和机制研究提供了一个经典的模型系统。为CAP开发的转录激活的机制描述比任何其他转录激活的例子的描述更接近于完整。然而，CAP介导的转录激活的完整结构基础仍不清楚。值得注意的是，近年来，CAP提供了一个很好的系统来研究构成变构基础的结构-和动力学-功能关系。CAP提供了第一个实验确定的系统，在该系统中，变构相互作用通过蛋白质运动的变化来调节，而蛋白质的平均结构没有变化。这个项目的主要目标是使用CAP作为一个模型系统来解决关于变构调节和转录激活的基本问题。综合的结构、动力学和热力学方法将用于(1)表征变构性质改变的CAP突变体的动力学及其与DNA的相互作用能量；(2)确定依赖于CAP的I类和II类启动子组件的溶液结构；以及(3)确定CAP介导的整个转录起始复合体组装的结构基础。广泛影响除了解决基本的生物学问题外，该项目还将用于培训学生结构生物学、生物物理学和分子生物学，这些领域正在迅速融入21世纪的科学。博士后、研究生和本科生将有机会参与一个多学科项目，该项目旨在开发突破性的方法，以便能够通过高分辨率核磁共振光谱来表征超分子蛋白质复合体的结构和动态特征。这将使研究人员能够从多学科和互动的角度处理问题，从而第一手体验到将最先进的方法应用于重要生物学问题的效用。将结构、动力学、热力学和动力学方法结合起来研究复杂蛋白质系统的范例将包括在目前由国际和平研究所设计的一门新课程中，以例证使用跨学科和定量方法来回答具有科学意义的问题的价值。该课程面向罗格斯大学分子生物科学、化学和化学生物学、生物医学工程和生物医学专业的研究生和高级本科生。