DOI
https://doi.org/10.25772/W1EV-6W44
Author ORCID Identifier
https://orcid.org/0000-0002-1296-1293
Defense Date
2021
Document Type
Thesis
Degree Name
Doctor of Philosophy
Department
Chemical Biology
First Advisor
T. Ashton Cropp
Second Advisor
Matthew C. Hartman
Third Advisor
Julio C. Alvarez
Fourth Advisor
Tomasz K. Kordula
Abstract
Most of the work vital for the survival of the cell are performed by proteins. The primary sequence of proteins is composed of 20 canonical amino acids. However, these 20 amino acids do not offer the full range of functionalities needed for proteins to do their job. Often times, post-translational modifications (PTM’s) occur to add the required functional group. This interaction between all the different proteins in the proteome in addition to PTM’s create a complex system that scientists are still trying to unravel to this day. Many different tools had been developed for this purpose, and one such tool is unnatural amino acid mutagenesis. The ability to site-specifically incorporate an unnatural amino acid with novel chemical and physical properties allowed scientists to probe and observe protein-protein interactions that had previously been difficult to perform.
This dissertation discusses work done to incorporate o-vinyl-tyrosine, 4-fluorohistidine, photocaged histidine, and Nε-L-thiaprolyl-L-lysine (ThzK) into proteins using the pyrrolysl-tRNA synthetase and tRNAPyl pair. Additionally, work to increase incorporation of UAA’s by increasing its cellular uptake is also discussed. Incorporation of o-vinyl-tyrosine to replace a catalytically active tyrosine residue allowed for control of the activity of an enzyme via chemical decaging using a tetrazine in an IEDDA reaction. Likewise, incorporation of a photocaged histidine also allowed for spatial and temporal control of enzyme activity via irradiation with 365 nm light. UAA’s are often not commercially available and synthesis of UAA’s could be costly. Thus, efforts were made to improve incorporation efficiency and lower the amount of UAA needed. We were able to tackle this problem by improving the cellular uptake of UAA’s. We were able to increase the lipophilicity of ThzK by masking the carboxylic acid with a methyl ester.
With the use of unnatural amino acid mutagenesis, we were also able to partially synthesize a chemically reactive polyubiquitin probe targeting K48 linkage specific deubiquitinases. We were able to synthesize a UbK48ThzK-48UbHis6 dimer where the ThzK could be deprotected to a cysteine. The 1,2 aminothiol moiety could then perform native chemical ligation with Ub1-75SR forming a trimer. Using this method, we could create ubiquitin linkages of our choosing, allowing us to probe for different enzymes within the complex ubiquitin signaling system that targets the different ubiquitin linkages.
The application of unnatural amino acid mutagenesis is endless, ranging from mechanistic probes to use in therapeutics. The work described in this thesis was able to increase the repertoire of available UAA’s and PylRS mutants that could be used as tools to improve understanding of the many different protein-protein interactions in the proteome.
Rights
© Jenny Cheung
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-13-2021