Structural and Dynamic Studies of Catabolite Activator Protein Complexes

智力优势遗传调控蛋白靶向基因组内的特定位点，增强或抑制转录活性以引起细胞反应。大肠杆菌分解代谢物激活蛋白（CAP，也称为cAMP受体蛋白，CRP）是一种通用的转录激活因子，可调节200多种基因的表达。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描述任何其他转录激活的例子都更接近完整。然而，cap介导的转录激活的完整结构基础仍然未知。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-和动态-功能关系。CAP提供了第一个通过实验鉴定的系统，其中变构相互作用是通过蛋白质运动的变化介导的，而蛋白质的平均结构没有变化。该项目的主要目标是使用CAP作为模型系统来解决有关变构调节和转录激活的基本问题。将采用综合的结构、动力学和热力学方法来(1)表征具有改变的变构特性的CAP突变体的动力学及其与DNA的相互作用能量；(2)确定I类和II类cap依赖启动子亚组件的溶液结构；(3)确定整个cap介导的转录起始复合物组装的结构基础。更广泛的影响除了解决基本的生物学问题外，该项目还将用于培养学生在结构生物学、生物物理学和分子生物学方面的知识，这些领域正在迅速成为21世纪科学的一部分。博士后、研究生和本科生将有机会参与一个多学科项目，旨在开发突破性的方法，通过高分辨率核磁共振波谱技术来实现超分子蛋白质复合物的结构和动态表征。这将使研究人员能够从多学科和互动的角度来处理问题，从而亲身体验将最先进的方法应用于重要的生物学问题的效用。结合结构、动态、热力学和动力学方法来研究复杂蛋白质系统的范例将包括在PI目前设计的新课程中，以举例说明使用跨学科和定量方法来回答科学重要性问题的价值。本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aps专业的研究生和高级本科生。

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.