Structural Studies of Fluorescence Lifetime Biosensors and Tunnel-Directed Inhibition of Human Dihydroorotate Dehydrogenase Restricted; Files Only

Abstract

This thesis applies structural biology, notably X-ray crystallography, to two distinct protein systems under a common goal: exploiting dynamic structure to understand function. The first part of the thesis explores genetically encoded fluorescent lifetime biosensors, focusing on the lactate sensor LiLac and the glucose sensor SweetieTS. Crystallization campaigns and structure-solution efforts are described for ligand-bound LiLac and SweetieTS constructs: for LiLac, the goal was to obtain a lactate-bound structure to pair with an existing lactate-free model; and for SweetieTS, the aim was to solve both ligand-free and glucose-bound states from crystals grown in the presence of glucose. These paired structures are used to propose how ligand-induced rearrangements in the protein scaffold could help explain observed changes in fluorescence lifetime. In the second part, the focus shifts to human dihydroorotate dehydrogenase (huDHODH), a mitochondrial enzyme that oxidizes dihydroorotate and passes electrons into the ubiquinone pool through a hydrophobic tunnel that also accommodates small-molecule inhibitors. Guided by prior Escherichia coli DHODH structures bound to the inhibitor HQNO, this work designs, expresses, and purifies a panel of huDHODH tunnel mutants intended to probe how specific tunnel residues affect HQNO binding and inhibition. While inhibitor studies of this panel are ongoing, the work presented here establishes the constructs and experimental workflow needed for future structure–function studies. Together, these projects show how a shared crystallographic toolkit can both clarify proposed mechanisms in biosensors and prepare the ground for systematic inhibitor studies on a clinically relevant enzyme.

Table of Contents

Chapter 1: Introduction 1

1.1 Structural Biology 1

1.2 Lifetime Biosensors 2

1.3 Role and Mechanism of Dihydroorotate Dehydrogenase 5

1.4 Relevant Techniques 8

1.4.1 Protein Expression 8

1.4.2 X-ray Crystallography 12

1.4.3 Inhibitor Studies 14

1.5 Research Themes 15

Chapter 2: Genetically Encoded Fluorescence Lifetime Biosensors 17

2.1 Background 17

2.1.1 Sensor Engineering and Evolution 17

2.1.2 SweetieTS 17

2.1.3 LiLac 18

2.2 Aims 20

2.3 Methods 20

2.3.1 SweetieTS 20

2.3.2 LiLac 22

2.4 Results/Discussion 23

2.4.1 SweetieTS 23

2.4.2 LiLac 30

Chapter 3: Human Dihydroorotate Dehydrogenase 34

3.1 Background 34

3.1.1 DHODH Structure 34

3.1.2 Therapeutic Relevance 34

3.1.3 HQNO and HAQs as Inhibitor Candidates 35

3.1.4 Structural Characterization and Prior Work in the Davis Lab 37

3.2 Aims 39

3.3 Methods 39

3.3.1 Site-Directed Mutagenesis 39

3.3.2 Transformation 43

3.3.3 Test Expression 43

3.3.4 Medium-Scale Expression 48

3.3.5 Purification 51

3.3.6 Large Scale Re-Expression and Purification 55

3.4 Results/Discussion 57

Conclusion 60

References 63

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biophysics, General Chemistry, Biochemistry Chemistry, Molecular
Keyword	HAQ Biosensors Structural Biology Biophysical Chemistry Protein Dihydroorotate Dehydrogenase DHODH Fluorescence Lifetime Hydroxy-alkylquinolines X-ray crystallography Enzyme Inhibitor
Committee Chair / Thesis Advisor	Katherine Davis, Emory University
Committee Members	Kate McKnelly, Emory University Antonio Brathwaite, Emory University

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