Synthesis and the Genetic Encoding of Novel Lysine Post-Translational Modifications

Author

Sahan A. Galbada Liyanage, Virginia Commonwealth UniversityFollow

DOI

https://doi.org/10.25772/WYE2-H078

Author ORCID Identifier

https://orcid.org/0000-0002-0166-0399

Defense Date

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Dr. T. Ashton Cropp

Abstract

Post-translational modifications (PTMs) play a significant role in regulating cell signaling and regulating the inner cellular environment. The study of PTMs and their primary biological functions is critical for the development of new medicines targeting the pathways and biomolecules responsible for the PTMs.

Previous studies revealed the direct coupling of the β -hydroxybutyryl lysine (K_BHB) modification with the gene expression. K_BHB in histone proteins links metabolism to epigenetics and gene expression and enables to study in-depth, how cell metabolism controls the gene expression and vice versa. Similar novel histone modification, K_Lac, attracts a special interest since lactic acid is generated highly in cancer cells due to the huge energy requirement. To study the PTM in-depth, it is critical to obtain homogeneously modified proteins. This study presents a methodology to obtain acylated lysine modified form of proteins in a homogenous composition.

This thesis reports the synthesis of N^ε-β-hydroxybutyryl lysine (K_BHB), K_Lac, and several other lysine PTMs. Synthesized lysine PTMs were screened using a small library of pyrrolysyl-tRNA Synthetase derived variants to find an enzyme that can recognize the lysine PTMs as substrates to aminoacylate the tRNA^Pyl_CUA. Finally, the discovered PylRS/tRNA^Pyl pair was used for the genetic incorporation of the synthesized lysine PTMs and encoding them into proteins as a response to the amber stop codon. This method enabled us to obtain a homogenous composition of proteins with the post-translationally modified form, which is focused site-specifically. This will greatly enable further studies directed at understanding the biological function of these PTMs.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-17-2020