Protein Structural Defects: Sites for Small Molecule Binding and Regulation

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This project will investigate novel experimental approaches to detect sites used by proteins to bind small molecule cofactors that are essential to their function. Such knowledge is critical to understanding how cells use protein-based molecular machines for many tasks, ranging from sensing changes in their microenvironment to controlling the speed of complex biochemical reactions inside of them. These protein machines often utilize small molecule cofactors to perform and control their various functions. Knowing how these cofactors bind their protein targets - and critically, where they do so on the protein - is essential for understanding how normal biological function is achieved, how this function is perturbed in disease, and how one might generate new regulatory molecules for biotechnology or medicine. This project will investigate the development of a novel approach to address where and how cofactors bind to proteins by taking advantage of pressurized water to help quickly probe for cofactor-binding sites, coupled with NMR spectroscopy to yield atomic precision. The successful completion of the planned research will yield new techniques which can be widely applied in academic and industrial laboratories, application results to a wide range of protein targets, and the training of students and researchers in a combination of cutting-edge biophysical and biochemical methods and analyses.This project combines multiple solution NMR approaches with complementary experimental and computational studies to investigate how the unusual structure and dynamics of cavities within proteins form sites suitable for small molecule binding and regulation. Such work is significant in providing quantitative descriptions of ligand-binding site properties, advancing the understanding of fundamental principles in biology and enabling the development of novel genetically-encoded sensors and ligand-activated proteins useful for the chemical and synthetic biology communities. This research program focuses on studying cavities within a large family of ligand-controlled protein/protein interaction domains, many of which have small, hydrated defects within their cores which can potentially serve as regulatory small-molecule binding sites. Several fundamental aspects of such binding remain cryptic at this time, most surrounding how ligand binding can occur despite these cavities being completely isolated from water. As well, there is almost no understanding of how protein dynamics - well-known to impact ligand specificity and the transmission of allosteric changes in many other systems - are involved in such protein-ligand interactions. To address these shortcomings, this project will utilize high pressure solution NMR spectroscopy in combination with other biophysical and biochemical approaches to: 1). Ascertain the prevalence of cavity prevalence and ligand binding within a subset of domains, 2). Determine how high pressure affects cavity structure and dynamics, allowing different conformations to be accessed with and without ligands and 3). Examine the functional linkage of ligand binding to control of effector domains.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.