Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Ernest J. Petersson

Abstract

Thioamides have been used for various applications with small molecules and peptides, including as protease sensors, fluorescence quenching probes, folding probes, and handles for site-specific chemical modification. However, their use in proteins has been limited due to cumbersome incorporation through semi-synthesis, as well as their unknown effects on protein structure. In this work, I address these issues from multiple perspectives. I present improvements for the synthesis of thioamide-containing peptides, which are needed for semi-synthesis. I also investigate the effects that thioamide substitutions have on tertiary structure in a model protein and show that based on thermal denaturation experiments, the position of thioamide substitution can have a significant impact on the tertiary fold. Furthermore, I demonstrate a new semi-synthetic strategy to obtain mg quantities of a thioamide-containing protein that will enable structural investigation by Nuclear Magnetic Resonance spectroscopy and X-Ray crystallography. Finally, I describe my attempts at the genetic incorporation of thioamides using a combination of unnatural amino acid mutagenesis and amber suppression. While the last part was not successful, the efforts to improve semi-synthesis of thioamides will enable the use of thioamides as probes in proteins in a similar fashion as they have been used previously in peptides.

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