Electronic Theses and Dissertations

Date

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

Committee Chair

Abby Parrill-Baker

Committee Member

Daniel Baker

Committee Member

Daniel Baker

Committee Member

Judith Cole

Committee Member

Bernie Daigle

Committee Member

Xiaohua Huang

Abstract

G-Protein-Coupled Receptors (GPCR) are membrane protein receptors involved in most eukaryotic signaling processes. GPCR are the largest pharmaceutically relevant membrane protein group, serving as targets for approximately one-third of all drugs currently approved by the FDA and many drug candidates presently undergoing clinical trials. Still, only about one-fourth of all known GPCR are current pharmaceutical targets, leaving vast untapped potential. Chapter 1 describes the conserved structure of GPCR, and briefly introduces their diverse ligands and functions. The pharmaceutical relevance of GPCR is highlighted, along with some of the challenges associated with their structure determination that have hindered our understanding of GPCR. Some of the computational methods for determining GPCR structures and their limitations are introduced. Chapter 2 first presents background information on the historic attempts to generate water-soluble membrane proteins and some of the early approaches toward achieving water-soluble GPCR. Chapter 2 then describes the methods for the research presented herein, which revolve around the application of the published QTY code to derive mutations sets designed to be transferable across class A GPCR. A C-terminal emerald green fluorescent protein (EmGFP) is used as a protein folding reporter to screen culture conditions for optimal protein expression in E. coli. Chapter 3 exhibits and discusses the results from the research conducted herein. The design of the informatic and tailored approaches to the QTY code application to the GPCR selected as proof of principle are described. Three designed variants each of D2, P2Y12, and CB1 were ordered, and four of the nine were successfully subcloned into the E.coli pET-28b (+) plasmid containing the EmGFP fusion protein. The conditions for protein expression were optimized through small-scale protein expression trials. GPCR constructs were purified from production-scale protein expression, resulting in the conformation of two constructs from the cleared soluble fraction of cells. Chapter 4 summarizes the research presented herein and briefly describes potential considerations for the continuation of this research. The alteration of Ni-NTA resin binding times and the addition of protease inhibitor cocktail to cleared soluble fractions from lysed cell pellets is proposed. Other suggested approaches include additional expression systems, ionic liquids to solubilize proteins, a method to reduce inclusion body formation, protein purification from insoluble fractions, fusion protein linker sequence optimization/ addition, and further development of the informatic application of QTY code mutations. Three appendices follow the body of this document. Appendix A contains two sections, separated into supplemental figures and tables referenced throughout the text. Appendix B describes additional research conducted that is intended to be a first author publication in the Journal of Molecular Graphics and Modeling. Appendix C describes published research conducted with collaborators at the University of Tennessee Health Science Center.

Comments

Data is provided by the student.”

Library Comment

Dissertation or thesis originally submitted to ProQuest

Notes

Open Access

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