Ligand binding studies of a plasmid encoded dihydrofolate reductase by fluorine NMR: towards quantifying ligand binding inside the cell

Author

Gabriel Jose Fuente Gomez, University of Tennessee, KnoxvilleFollow

Date of Award

8-2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Gladys Alexandre

Committee Members

Dan Roberts, Francisco Barrera, Engin Serpersu

Abstract

Antibiotic resistance is a worldwide problem. The excessive use and misuse of antibiotics have resulted in the development of drug resistance. One example involves the antibiotic trimethoprim (TMP). TMP selectively inhibits the bacterial enzyme dihydrofolate reductase (DHFR) over its human counterpart. Inhibition of DHFR results in inhibition of DNA synthesis and ultimately in cell death. Unfortunately, extensive use and dissemination of TMP over the last forty years has resulted in the development of drug resistance in the form of highly transmissible plasmids. R67 dihydrofolate reductase is a plasmid-encoded enzyme that confer resistance to TMP. Our lab has studied how R67 DHFR works in vitro using diluted conditions. However, most proteins function inside cells under crowded and heterogeneous environment. As ¹⁹F labeled proteins in complex mixtures can be characterized by NMR, we report our initial expression and characterization of R67 DHFR. To obtain such information, we have labeled R67 DHFR with fluorine at different positions in the indole ring and showed that fluorine incorporation into R67 DHFR does not affect the structure and function of the protein. We have characterized the ¹⁹F NMR spectra of apo R67 DHFR and optimized fluorine incorporation. Furthermore, fluorinated R67 DHFR shows different signals for binding of cofactor, NADP+ , and substrate, dihydrofolate (DHF). We have obtained similar dissociation constants for the cofactor and substrate of R67 DHFR by both NMR and ITC. In our next step, we will measure dissociation constants in more complex solutions. We envision using fluorinated protein to quantify the effect of osmolytes and crowders on the binding affinity of substrate and cofactor towards R67 DHFR.

Recommended Citation

Fuente Gomez, Gabriel Jose, "Ligand binding studies of a plasmid encoded dihydrofolate reductase by fluorine NMR: towards quantifying ligand binding inside the cell. " PhD diss., University of Tennessee, 2020.
https://trace.tennessee.edu/utk_graddiss/9910