Research Objectives

Proteins carry out a myriad of tasks in the body, such as providing structure and motion to our bodies, transporting oxygen in our blood, serving as antibodies to protect us against invading bacteria, and causing reactions that are involved in all aspects of metabolism to go faster. Those proteins that catalyze reactions, i.e. make them go faster, are specifically called enzymes. My research group is interested in learning how these enzymes are regulated or, in other words, constantly turned "on" and "off" to control metabolism. For example, following a large meal containing an abundance of sugars, certain enzymes need to be turned "on" in order for the body to break down the sugars for energy; when sugars are consequently depleted and the body needs to start using other substances for energy, these same enzymes may need to be turned "off". The control mechanism or "on/off" switch in many enzymes involves a change in the shape, i.e. conformation, of the enzyme in response to the binding of metabolites that might be accumulating under a given set of metabolic conditions. The change in conformation of the enzyme, termed an allosteric change, results in the enzyme being better able (turned "on") or less able (turned "off") to carry out its function. Exactly how the binding of metabolites in one area of the protein causes the enzyme to alter its global conformation and how these changes in shape specifically affect its ability to function are all questions we attempt to address in this research.

An understanding of the molecular basis for allostery is integral to any biomedical research targeting cellular regulation. Carbamoyl phosphate synthetase (CPS) from E. coli serves as our model system for examining such allosteric mechanisms. CPS catalyzes the production of carbamoyl phosphate for the formation of pyrimidine and arginine during nucleotide and protein biosynthesis, respectively, as well as for the detoxification and excretion of ammonia in ureotelic organisms. Although much research has been directed to understanding its action and regulation, broad questions remain. What regions of the protein matrix serve as conduits of communication between the allosteric, catalytic, and oligomerization domains? How do regulator molecules specifically influence each reaction center? How are the three separate reaction centers synchronized in the production of carbamoyl phosphate, i.e. are allosteric communications manifested between substrates themselves within different active sites? Do allosteric couplings (the nature and magnitude of the impact that one ligand imparts on the binding affinity of a second ligand) change in response to oligomerization into the tetrameric species that predominate in vivo? To address these questions, we will develop fluorescence probes for directly monitoring allosteric communications within CPS. Changes in the steady-state and dynamic properties all such fluorophores will be systematically monitored as a function of ligand binding to highlight regions of the protein matrix specifically responsive to allosteric transitions. Allosteric couplings between various ligand combinations will then be individually isolated and quantified via observed fluorescence changes by applying a “linked-function” analysis, resolving the matrix of allosteric communications between the 3 active sites and 2 modulator sites. Finally, couplings, once identified and characterized, will be monitored as a function of protein concentration to assess the potential role of oligomerization on the allosteric regulation and/or synchronization of E. coi CPS.