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Title page for ETD etd-03252019-091018

Type of Document Master's Thesis
Author Metts, Adam Mason
Author's Email Address adam.m.metts@vanderbilt.edu
URN etd-03252019-091018
Title Synthesis and Validation of a Trifunctional Trimethoprim-based Probe for Use with Degradation Domain System
Degree Master of Science
Department Chemistry
Advisory Committee
Advisor Name Title
Lars Plate Committee Chair
Brian O. Bachmann Committee Member
  • chemical biology
  • proteomics
Date of Defense 2019-03-25
Availability restricted
The cell is regulated by a vast network of dynamic protein interactions that facilitate various sub-cellular functions. To systematically monitor and characterize these transient and rapidly changing protein interactions, new tools are needed to track these processes with spatial and temporal resolution. Here, we describe our progress toward designing a chemical biology tool to rapidly induce protein expression and simultaneously allow isolation of the protein of interest. The work entails the synthesis and validation of a trifunctional small-molecule probe for use with a modified degradation domain system. The probe was designed to 1) provide ligand-induced stability to the degradation domain, 2) bind covalently to the degradation domain, and 3) contain a handle for Click-Chemistry modification to allow for pull-down of the bound protein complex. According to these design principles, we synthesized several trifunctional, trimethoprim-based stabilizing ligands that include an alkyne Click-Chemistry handle and a Cys-reactive electrophile to form covalent adducts with residues near the DHFR active site. For proof-of-principle, we employed destabilized dihydrofolate reductase (ddDHFR) fused to yellow fluorescent protein (YFP). We engineered mutants of this ddDHFR-YFP complex to include active site cysteine residues to screen for reactivity with the probe. We used live-cell fluorescence imaging to confirm that the synthetic probe stabilizes the DHFR complex, and we used a Cu(I)-mediated Click reaction to confirm that the probe binds covalently and specifically to the DHFR complex. In a future application, our tool has the potential to be used for time-resolved interactomic profiling.
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