Andrew Lee, PhD, an associate professor at the UNC School of Pharmacy, has received a grant from the National Institutes of Health for a collaboration with fellow School faculty Scott Singleton, PhD, to study at how protein motions affect function and inhibition in the enzyme dihydrofolate reductase.
The grant is titled “Intra- and Intermolecular Dynamics of Dihydrofolate Reductase” and is worth more than $1 million over four years.
“Proteins are dynamic molecules, and this aspect often contributes to their function in binding ligands and catalyzing reactions,” Lee says. “However, the traditional methods used to determine molecular structures don’t directly report on the extent and timescales of fluctuations of the protein.”
Lee and Singleton, both faculty members in the Division of Medicinal Chemistry and Natural Products, will try to study those aspects of protein fluctuation through NMR relaxation experiments, chemical synthesis, and enzyme kinetics. The relaxation experiments allow researchers to determine the flexibility for atoms in a protein molecule, information that Lee says is not typically obtained from traditional methods.
The collaboration will draw upon Lee’s expertise in NMR spectroscopy and Singleton’s experience in chemical synthesis and transient- and pre-steady-state enzyme kinetics—the study of the reaction that takes place in the few milliseconds just after an enzyme is mixed with a substrate and before the product is formed.
Lee and Singleton will use small drug or drug-like molecules to see how drugs with different binding affinities affect the internal motions of DHFR in E. coli. They will also use synthetic chemistry to tone down the binding abilities of compounds. The researchers will then use these poorly binding compounds to help them observe what motions may occur in the protein to help break the bond between the compound molecules and the DHFR.
DHFR is a key enzyme for cells. One of its functions is converting folic acid from a vitamin form into a form usable in biosynthetic pathways, which is important for making many of the constituents of DNA and other protein amino acids. It is a well-known enzyme in the research realm and a target of anticancer, antibacterial, and antifungal drugs.
“DHFR also happens to be the poster child of small dynamic proteins or enzymes,” Lee says. “We are using it as a model system to study how the event of drug binding alters the dynamic nature of proteins, as little work has been done in this area.”