Proteins function by greatly accelerating specific chemical reactions (catalysis), binding to proteins and other ligands, as well as via other mechanisms; these actions are essential for all life processes. The physical mechanisms used for enzyme catalysis, protein regulation, and protein interactions, are fundamental to understanding protein function. Addressing these physical mechanisms at the deepest level, requires understanding basic forces and the interplay of structure, dynamics, and energetics within proteins. Thus, such understanding will have widespread and profound consequences. As understanding these underlying forces and the resulting macromolecular behaviors has been enormously challenging and will require multidisciplinary, highly integrated approaches, this project involves a multidisciplinary team with broad and deep expertise that allows the use, integration, and innovation of approaches encompassing structure-function and energetic probes. This proposal will aid in training of a diverse and highly impactful group of young scientists with scientific rigor and integrity. This project will also implement a novel, highly impactful integration of research between graduate research universities and underrepresented minority-rich undergraduate institutions, synergistically enhancing research and mentoring.
This research focuses on the enzyme ketosteroid isomerase (KSI) as an exceptionally powerful system to pose and dissect fundamental questions in protein function and energetics, and will use KSI and other protein and model systems to understand hydrogen bond structure and energetics and to determine the interplay of structure, dynamics, and energetics throughout a protein scaffold. The hydrogen bond may be the most fundamental interaction in biology and yet our understanding of and ability to predict the structures, energetics, and dynamics of hydrogen bonds and hydrogen bond networks are remarkably limited. In this research a quantitative and predictive model for hydrogen bond structure will be developed that focuses on factors that determine hydrogen bond length and coupling; hydrogen bond energetics within protein environments. The wide array of approaches utilized include site-directed mutagenesis and unnatural amino acid incorporation; protein structure and dynamics via NMR and cryo- and room temperature X-ray, and neutron crystallography; atomic-level energetics, dynamics, and catalysis via quantum mechanics/molecular mechanics (QM/MM); and assessment of small molecule hydrogen bond (H bond) energetics and ligand association via 1H NMR, isothermal titration calorimetry, small molecule neutron and X-ray diffraction, and quantum mechanics. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.
