Structural and Dynamic Studies of Catabolite Activator Protein Complexes

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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.