Charalampos G. Kalodimos - US grants

This grant partially supports the acquisition of an 800 MHz NMR cryoprobe to be sited at Rutgers University, Busch science campus. The cryoprobe technology will initially serve a minimum of four New Jersey institutions including Rutgers-New Brunswick (NB), Rutgers-Newark, Princeton University and Montclair State University (MSU). Eleven faculty members, three from Rutgers-NB/RWJ, two from Rutgers-Newark, four from Princeton and two from MSU are participating in the New Jersey Wide 800 MHz NMR Facility. Hands-on NMR experiments will be performed by approximately 60 graduate students and postdocs, as well as undergraduates involved in NMR research. The NMR facility will also have significant impact on research and teaching of additional faculty members in New Jersey through the research collaborations with the faculty and students in the facility.

The research activities of the users of the facility are in molecular biophysics with emphasis in the following areas: Protein structure determination; Conformational studies and dynamics of folded and unfolded proteins to understand the basis of protein folding and misfolding and their associated medical disorders; Studies of nucleic acid structure and interactions; NMR investigations of protein-protein and protein-nucleic acid interactions; Design and informational analysis of small bio-medically important peptides; and NMR methods development for next generation application.

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.

Intellectual merit: The objective of this proposal is to use solution NMR spectroscopy to study RNAP in various transcriptional stages. The DNA-dependent RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation in all cellular organisms. RNAP is a remarkable protein machinery capable of (i) specifically binding to promoter sites along the DNA, (ii) melting the double-stranded DNA (dsDNA) to form the transcription "bubble", and (iii) synthesizing the RNA chain complementary to the DNA template strand using nucleoside triphosphate (NTP) substrates. The essential core component of the bacterial RNAP (subunit composition alpha 2, beta, beta prime and omega) has been evolutionarily conserved from bacteria to humans. Sequence conservation points to structural and functional homologies, rendering the simpler bacterial RNAPs excellent model systems for understanding the basic principles at work for all cellular RNAPs. Recent progress in the structural, biochemical and biophysical characterization of RNAP has highlighted this enzyme as a complex, multifunctional protein machinery that functions by using an intricate balance of structural and dynamic changes.
The goal of this project is to obtain integrated structural and dynamic information of the intricate mechanisms that underpin RNAP functionality by studying global and subtle structural changes as well as the amplitude and the time scale of functional motions. Towards this goal, the project will (1) develop strategies and methodologies to overcome the large size and complexity of RNAP, and (2) acquire structural and dynamic data on RNAP by applying an integrated NMR approach involving experiments tailored for (i) obtaining long-range structural information, (ii) detecting transiently populated conformational states, and (iii) determining both fast and slow time-scale motions and assessing their significance.
Broader impact: Accomplishment of these objectives will have a tremendous impact on various fields. First, it will provide site-specific structural and dynamic information of RNAP during its action in solution, thereby offering unprecedented insight into the functional mechanisms of this important enzyme. Second, it will provide a model study about how NMR can be applied to obtain integrated structural and dynamic information on supramolecular biological systems. Third, it will establish NMR as a powerful tool for the dynamic characterization of large, intricate protein machineries by complementing static structures offered by X-ray crystallography.
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 characterization of supramolecular protein complexes by high resolution NMR spectroscopy. This will enable participants in the project 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. Two graduate students, supported by training grants and teaching assistantships, will do their theses on this project. They will be involved in the development of new labeling protocols and in the application of advanced NMR methodologies towards the dynamic and structural characterization of RNAP. 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 and biomedical importance. The course is intended for a large, diverse audience consisting of graduate and advanced undergraduate students in the programs of Molecular Biosciences, Chemistry & Chemical Biology, Biomedical Engineering and BIOMAPS at Rutgers University.

Intellectual merit
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.