Toward the Probing of DHQS Activity by Protein Engineering through the Introduction of Unnatural Amino Acids and the Selection of tRNA/tRNA Synthetase Pairs

Authors

Shaina E. Ives, University of Nebraska-LincolnFollow

Date of this Version

12-2015

Document Type

Article

Citation

Ives, S. E. Toward the Probing of DHQS Activity by Protein Engineering through the Introduction of Unnatural Amino Acids and the Selection of tRNA/tRNA Synthetase Pairs. University of Nebraska-Lincoln. 2015.

Comments

A Thesis Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemistry, Under the Supervision of Professor Jiantao Guo. Lincoln, Nebraska: December 2015

Copyright (c) 2015 Shaina E. Ives

Abstract

Protein engineering is a valuable tool that allows scientist to explore how an enzyme works by mutation of key residues. This method has been used to improve function or stability of enzymes, thus allowing their use in both the lab and in industry to be expanded. Genetic incorporation of unnatural amino acids (unAA) can be used with protein engineering to exceed the current limitations, due to the limited number of functional groups of the 20 common amino acids.

The majority of this thesis will discuss the progress on incorporating the various unAA into the active site of the enzyme, Dehydroquinate Synthase (DHQS). Previously this enzyme has been understood through the use of inhibitors as well as crystallization of the enzyme with its substrate and cofactors. This work begins to explore the chemical reactions of this enzyme catalysis, as well as enhancing the known tools of genetic incorporation of unAA. This masters thesis focuses on my work the in: (1) the synthesis of the unAAs including hydroxyquinolin-alanine and 2-(5-carboxythienyl)alanine; (2) the identification of aminoacyl tRNA synthetase recognizing 2-(5-bromothienyl)alanine, 2-mercapto-L-histine, diiodo-histine, and 2-(5-carboxythienyl)alanine; and (3) the incorporation of hydroxyquinolin-alanine and 2-(5-bromothienyl)alanine into DHQS and the evaluation of the resulting mutants. I also discuss briefly the attempted synthesis of photo-threonine.

Advisor: Jiantao Guo