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

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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.