Development Of Thioamides As Protein Probes

Loading...
Thumbnail Image
Degree type
Doctor of Philosophy (PhD)
Graduate group
Biochemistry & Molecular Biophysics
Discipline
Subject
Native Chemical Ligation
Protein Stability
Thioamide
Biochemistry
Cell Biology
Organic Chemistry
Funder
Grant number
License
Copyright date
2020-02-07T20:19:00-08:00
Distributor
Related resources
Contributor
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.

Advisor
Ernest J. Petersson
Date of degree
2019-01-01
Date Range for Data Collection (Start Date)
Date Range for Data Collection (End Date)
Digital Object Identifier
Series name and number
Volume number
Issue number
Publisher
Publisher DOI
Journal Issue
Comments
Recommended citation