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
Included in
Biochemistry Commons, Microbiology Commons, Molecular Biology Commons, Organic Chemistry Commons