Structural and Dynamic Studies of Allosteric Activation of the Catabolite Activator Protein

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。这种调节机制的基本特征是各种配体的多个结合位点的存在以及这些结合位点之间的通信，这些结合位点通常位于许多埃之间。这个项目的目标是解决两个基本问题：第一，调节蛋白是如何通过变构效应结合切换到激活/抑制状态的？第二，在蛋白质中调节协同性的机制是什么？将采用核磁共振、等温滴定量热法（ITC）和蛋白质工程的综合方法，在原子分辨率上表征分解代谢物激活蛋白（CAP）变构活化的结构、动力学和能量机制。为了深入了解活化过程，我们设计了具体的目标，以提供CAP在载脂蛋白状态下及其与cAMP和DNA相互作用的结构和动态基础。此外，该研究将包括基因鉴定的突变体，这些突变体改变了蛋白质的变构特性，为研究替代变构途径提供了良好的系统。了解CAP的变构激活过程将具有普遍的重要性，因为数百个编码CAP相关蛋白的基因正在被识别。该项目将为学生提供核磁共振、结构生物学、热力学和各种生物物理技术方面的培训。该项目还将加强位于纽瓦克的所有学院的教学基础设施。PI已经开发并引入了一门新的核磁共振波谱课程，他将利用从这项研究中获得的知识来设计一门课程，重点是生物分子的相互作用和识别，结构和动力学在蛋白质功能中的作用，以及细胞过程的变构调节。

Genetic regulatory proteins target specific sites within the genome and either enhance or repress transcriptional activity to elicit cellular responses. The basic features of such a mechanism of regulation are the presence of multiple binding sites for various ligands and communication between these binding sites, which often are situated many angstroms apart. The objective of this project is to address two fundamental questions: First, how is a regulatory protein switched to its activated/suppressed state by allosteric effector binding? Second, what are the mechanisms that mediate cooperativity in proteins? An integrated approach using NMR, isothermal titration calorimetry (ITC), and protein engineering will be employed to characterize at atomic resolution the structural, dynamic, and energetic mechanisms that underlie the allosteric activation of the catabolite activator protein (CAP). To gain a detailed insight into the activation process, the specific aims have been designed to provide the structural and dynamic basis of CAP in its apo state and its interaction with cAMP and DNA. Moreover, the study will include genetically identified mutants that alter the allosteric properties of the protein providing excellent systems to study alternate allosteric pathways. Understanding the process of allosteric activation of CAP will be of general importance because hundreds of genes encoding CAP-related proteins are being identified. This project will provide training to students in NMR, structural biology, thermodynamics, and a variety of biophysical techniques. The project will also enhance the teaching infrastructure in all Institutes located at Newark. A new course in NMR spectroscopy has been developed and introduced by the PI, who will use knowledge obtained from this research to design a course focusing on biomolecular interactions and recognition, role of structure and dynamics in protein function, and allosteric regulation of cellular processes.