Genetic 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 impact In 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.

Project Report

Accumulating evidence indicates that internal dynamics can mediate allosteric interactions and modulate ligand binding. It is now well accepted that it is the inextricable link between structure and dynamics that ultimately control protein activity. However, how the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is not possible to rationalize solely on the basis of structural data. Although internal motions of proteins appear to have a major role in regulating protein activity, the nature of their contributions remains elusive, especially in quantitative terms. Over the last few years, our lab has reported novel and exciting findings about protein activity regulatory mechanisms through modulation of internal dynamics on different timescales. We have exploited the favorable properties of the catabolite activator protein (CAP), a prototype for allosterically regulated proteins, and used atomic- resolution NMR spectroscopy to characterize the structure and dynamics of several CAP variants with modified allosteric features. We have shown that changes in conformational entropy can determine whether protein–ligand interactions will occur, even among protein complexes with identical binding interfaces. We have further shown that that allosteric inhibition can be effected by destabilizing a low-populated conformational state that serves as an on-pathway intermediate for ligand binding, without altering the protein’s ground-state structure. The findings are of extreme importance as they, collectively, demonstrate how changes in fast internal dynamics (conformational entropy) and slow internal dynamics (energetically excited conformational states) can regulate binding activity in a way that cannot be predicted on the basis of the protein’s ground-state structure. In this project we used an integrated structural, dynamic and thermodynamic approach to study biological questions of fundamental interest and importance. Postdocs, graduate and undergraduate students that participate in the project were involved in the development of the methodologies that enabled characterization of large protein complexes by high resolution NMR spectroscopy. They were also involved in studying dynamic properties and understanding the often elusive role of protein dynamics in protein function. The complimentary techniques enabled our people 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.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
1121896
Program Officer
Michele McGuirl
Project Start
Project End
Budget Start
2011-08-01
Budget End
2014-07-31
Support Year
Fiscal Year
2011
Total Cost
$622,186
Indirect Cost
Name
Rutgers University
Department
Type
DUNS #
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901