Project Summary We propose to study the relationship between the structure, dynamics, and function of enzymes by examining how changes to their conformational ensembles regulate their catalytic functions. Understanding this relationship is critical for understanding macromolecular phenomena such as allosteric regulation, yet it remains difficult, because the relevant conformational changes involve a hierarchy of motions that occur across broad lengthscales (sub-Å to multi-nm) and timescales (ps-s). Our lab is developing a new generation of structural measurements that combine temperature perturbations with static and time-resolved X-ray crystallography, allowing us to explore the conformational landscapes of protein molecules in detail. We aim to apply these methods to temperature-sensitive orthologs from key enzyme families, including kinases, proteases, and ATP-dependent chaperones, to understand how changes to their conformational ensembles modulate their biological functions. The specific goals of our work are: (1) Use multi- temperature X-ray crystallography, combined with traditional biochemical and biophysical assays, to quantify the relationship between conformational states and catalytic activity. (2) Characterize previously invisible conformational states of enzymes, including cryptic pockets that can be targeted for drug discovery, using time-resolved temperature-jump crystallography. (3) Continue developing new hardware and software to improve the collection and analysis of data from multi-temperature and temperature-jump crystallography. Our research represents a novel approach to understanding how the balance of active and inactive conformations drives the regulation of protein function. Successful completion will yield new information about the structure-function relationships of biologically and clinically important enzymes and provide new opportunities for targeting them with therapeutics. We expect that similar changes to protein conformational ensembles underlie thermal regulation and other types of allosteric regulation in these enzyme families, and therefore we expect our results to be generally useful in understanding allosteric regulation more broadly. Finally, our work will develop a framework for studying the relationship between protein structure, dynamics, and function that exploits the response of protein conformational ensembles to temperature, and we aim to democratize the use of multi-temperature and temperature-jump crystallography as a general tool for the structural biology community.

Project Narrative The molecular motions of proteins underlie many important biological phenomena, such as allostery, yet they remain challenging to study at high spatial and temporal resolution. The goal of the proposed research is to implement a novel approach that combines temperature perturbations with structural, biochemical, and biophysical measurements to provide insight into the allosteric regulation of protein molecules that have key functions in health and disease. The knowledge generated from these studies will deepen our understanding of human biology and provide information that is actionable for drug discovery.