Electronic Theses and Dissertations

Identifier

6037

Date

2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Concentration

Biochemistry

Committee Chair

Abby L Parrill

Committee Member

Daniel L Baker

Committee Member

Ramin Homayouni

Committee Member

Judith A Cole

Abstract

G protein-coupled receptors (GPCR) comprise a superfamily of ~800 proteins that initiate diverse biological responses, leading to interest in GPCR as prospective pharmaceutical targets. These receptors share structural characteristics that include three extracellular loops, seven-transmembrane spanning alpha-helical segments, and three intracellular loops. While the first GPCR structure was solved by x-ray crystallography in 2000, less than 50 different GPCR have been crystallized thus far. Chapter 1 describes progress in GPCR structure determination since the crystallization of rhodopsin in 2000. An overview of technological advancements that enabled three-dimentional characterization of over 40 individual GPCR family members is further described. A comprehensive list of over 180 individual GPCR structures, including different GPCR functional states crystallized with agonists, antagonists, etc., is presented along with a comparative analysis of GPCR structure. The goal of Chapter 2 was to produce a water-soluble design that is transferrable throughout the GPCR family, producing ligand-binding functions comparable to the native protein. The adrenergicβ2 receptor(ADRβ2) served as the proof-of-principle for the water-soluble design. Mutations were introduced onto the surface of the protein to encourage the production of a water-soluble mimic. Mutants have been expressed in E. coli and purified at a yield of ~3 mg/mL. Functional assessment included circular dichroism, differential scanning calorimetry, and isothermal titration calorimetry. LC-MS/MS experiments following crystallization trials revealed the bacterial chaperone, GroEL. An alternative expression method to avoid GroEL expression is needed in order to characterize the water-soluble mimic. The work in Chapter 3 identified patterns of ligand-residue interactions in the GPCR family. Protein-Ligand Interaction Fingerprints (PLIF) was used to analyze 158 different GPCR crystal structures. A total of 44 sites were identified in the analysis. A subset of these sites displayed a variety of interaction types, indicative of a role in ligand selectivity. An alternate set showed consistent interaction strength, indicative of a role in binding affinity. Residue 6.48 plays little role in ligand binding, but is part of the "Trp Rotomer Switch" that exhibits sidechain conformational changes upon activation, as reflected in 17 ADRβ2 crystal structures.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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