Defense Date

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Thomas A. Cropp

Second Advisor

Katharine Tibbetts

Third Advisor

Katherine Belecki

Fourth Advisor

Fernando Tenjo

Abstract

Histidine is one of the most common catalytic residues found in proteins due to its unique imidazole moiety. The capability to incorporate caged histidine molecules into proteins selectively would therefore enable gain-of-function manipulation of mutant proteins by decaging histidines incorporated at positions critical for protein function. Proteinogenic incorporation of caged histidines selectively can be accomplished through the use of genetic code expansion; more specifically, with stop codon suppression to incorporate synthetic amino acids in vivo. The advancement in recent years of genetic code expansion has opened the doors to new worlds of peptide synthesis technologies with a growing list of non-canonical, or “unnatural” amino acids (UAAs) that are able to be incorporated into proteins. This dissertation records research conducted to contribute to the study of genetic code expansion, specifically in eukaryotic organisms. Herein I describe the development of multiple amber stop suppressor mutant aminoacyl-tRNA synthetase (aaRS)/tRNA systems in yeast capable of efficient and specific incorporation of a series of UAAs. These include, among others, a novel histidine UAA analogue synthesized for use in protein caging. In addition, I report the synthesis of novel decaging compounds designed to target this UAA with improved de-caging kinetics, based on the fast and bioorthogonal Inverse Electron Demand Diels-Alder (IEDDA) reaction. These novel UAAs and new promiscuous synthetase systems represent a significant expansion of the structural diversity of amino acids that can be incorporated into yeast proteins, and a basis for the development of a generalizable method for blocking and rescuing catalytic histidine interactions in vivo.

Rights

© Aidan Haney

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-9-2024

Available for download on Wednesday, May 09, 2029

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