Date of Award
Master of Science (MS)
College of Science and Mathematics
Chemistry and Biochemistry
Thesis Sponsor/Dissertation Chair/Project Chair
Nina M. Goodey
The goal of this project was to study the effect of the presence or absence of the cofactor NADPH on the binding and release of ligand methotrexate (MTX) to/from Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR). A previously developed, fluorescently-labeled Bs DHFR (C73A/S131CMDCC DHFR) was used to investigate the kinetics and protein conformational motions associated with the binding of NADPH and the binding of methotrexate to the holoenzyme. This Bs DHFR contains a distal cysteine where the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) can be covalently attached. This probe is sensitive to the local molecular environment, reporting on changes in the protein conformation associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) was also used to detect changes in conformational motion upon ligand and cofactor association and dissociation. Previous stopped-flow data indicates the presence of two native state Bs DHFR conformers that bind to ligand at different rates. Similarly, two conformations of Escherichia coli DHFR in unbound state were reported. Intrinsic tryptophan fluorescence of C73A/S131C Bs DHFR and probe fluorescence of C73A/S131CMDCC Bs DHFR both report on ligand binding. The labeled and unlabeled DHFRs report on the two different conformers, respectively. This study shows that NADPH binding significantly slows down the dissociation rate (approx. 1000-fold) of methotrexate from Bs DHFR from 0.015 ±0.007965 s-1 to 0.000021 ±0.00000271 s-1. This demonstrates the importance of NAHPH in the analysis and study of DHFR.
Foley, Ryan Seamus, "Presence of Cofactor NADPH Slows Down the Dissociation of Methotrexate from Dihydrofolate Reductase from Bacillus stearothermophilus" (2018). Theses, Dissertations and Culminating Projects. 102.