Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase Public Deposited

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  • March 21, 2019
Creator
  • Mauldin, Randall Vernon
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • Long-range amino acid communication is crucial for biological processes such as protein signaling, enzyme catalysis and allosteric regulation. In this work, the underlying dynamic networks that links distal regions of a protein to its catalytic center are illuminated, using dihydrofolate reductase (DHFR) as a model system. DHFR catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. Extensive biochemical analyses have established key regions that, upon mutation, mimic allosteric regulation by altering the rate of catalysis. To gain insight into the role that dynamics play in regulation, NMR spectroscopy is employed to measure protein motion on timescales ranging from picoseconds to microseconds. The dynamics of DHFR are perturbed in two ways: by binding high affinity inhibitors such as methotrexate and trimethoprim to the active site, or by mutating distal residues that reduce the catalytic rate by changing the dynamics of hydride transfer. These methods in conjunction allow us to track the dynamic response throughout DHFR as the perturbation originates from, or connects to, the active site. Tight binding ligands decouple global micro- to millisecond conformational switching, while altering the sub-nanosecond backbone and side-chain dynamics throughout the protein. Furthermore, distal mutations alter the dynamics within the active site and at regions of the protein known to be hydride transfer. These surprising results support the conclusion that the active site is functionally linked to distal regions of DHFR by discrete dynamic interactions, and that altering these interactions may be a mechanism to allosterically regulate catalysis.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics."
Advisor
  • Lee, Andrew
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  • Chapel Hill, NC
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  • Open access
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