Effect of Site-Specific Heating on Enzyme Catalysis Public

Abstract

Enzymes are nature’s catalysts. All enzymes have an energy landscape, and they lower those activation barriers to accelerate rates under biological temperature, pH, and concentrations. Enzymes have both static and dynamic components. Static “snapshots” of enzymes can be obtained with x-ray crystallography or cryo-electron microscopy to observe how the enzyme is folded, but a structural component alone cannot predict how enzymes move during catalysis. Although the contribution of protein motions to enzymatic catalysis has been heavily studied, experimental evidence reporting on the exact role of enzyme dynamics in catalysis is lacking. We seek to understand if dynamic motions in enzymes during catalysis represent preferred energy pathways. To interrogate the connection between enzyme catalytic motions and preferred energy pathways, dihydrofolate reductase (DHFR), which has a known network of coupled motions, is conjugated to gold nanoparticles (AuNPs). Enzyme activity is monitored as a function of the protein attachment site (distance to/from the network of coupled motions) on the AuNP, as well as of the timescale of laser pulsing. DHFR activity when attached to the AuNP close to the network (on the FG loop) is accelerated when excited by the pulsed lasers. When attached near the cofactor binding site network residue E101 (on an Alpha Helix), turnover is accelerated to a lesser extent. There is a greater degree of acceleration with fs pulsed laser than with ns pulses in both mutants. There is no rate acceleration when the AuNP is attached to DHFR away from the network (Distal Mutant) or via the histidine tag (also away from the network). There is no rate acceleration observed for any DHFR-AuNP attachment site with the continuous wave excitation. When the excitation timescale is fast enough, the heat flow into the protein affects the enzyme motions in catalysis, which are likely the motions involved in the search for reactive conformations, and the heat eventually thermalizes after these motions take place. This dissertation demonstrates a useful methodology for studying protein motions in enzyme catalysis, allowing us to investigate energy pathways in catalysis.

Table of Contents

Chapter 1 – Introduction 1

Section 1-1: Enzyme Dynamics, Catalysis, and Motions 1

Section 1-2: Dihydrofolate Reductase 5

Section 1-2.1: Overview of Enzyme and Function 5

Section 1-2.2: Catalytic Cycle 8

Section 1-2.3: Networks of Coupled Motions 11

Section 1-3: Gold Nanoparticles 18

Section 1-4: Bioconjugation of Proteins and Gold Nanoparticles 21

Section 1-4.1: Methods of Bioconjugation 21

Section 1-4.2: Characterization of Protein-Gold Nanoparticle Conjugates 22

Section 1-5: Energy Flow from AuNPs to Proteins Through Photoexcitation 24

Section 1-6: Chapter 1 References 27

Chapter 2 – Experimental Methodology 37

Section 2-1: Introduction 37

Section 2-2: Dihydrofolate Reductase Production 38

Section 2-2.1: Mutants of Dihydrofolate Reductase and Plasmid Design 38

Section 2-2.3: DHFR Expression and Purification 41

Section 2-2.4: TEV Protease Expression and Purification 42

Section 2-3: Materials for Protein-Gold Nanoparticle Conjugation 44

Section 2-3.1: Buffers 44

Section 2-3.2: Protein Stocks 44

Section 2-3.3: Gold Nanoparticle Synthesis 44

Section 2-3.4: Fluorescence Assay Components 45

Section 2-4: Conjugating Proteins to Gold Nanoparticles 46

Section 2-4.1: TEV Cleavage 46

Section 2-4.2: Protein-Gold Nanoparticle Binding Process 50

Section 2-4.3: Separation of Free Protein from Conjugates 51

Section 2-5: Analytical Methods for Characterization 51

Section 2-5.1: SDS-PAGE 51

Section 2-5.2: UV/Vis Absorption Spectroscopy 52

Section 2-5.3: Dynamic Light Scattering 52

Section 2-5.4: Fluorescence Assay to Determine Protein Concentration 53

Section 2-5.5: Transmission Electron Microscopy 54

Section 2-6: Activity Assays 54

Section 2-6.1: Standard Activity Assays 54

Section 2-6.2: Temperature Dependent Kinetics 55

Section 2-6.3: Lasers for Heating Experiments 55

Section 2-6.4: Light Driven Activity Assays 56

Section 2-7: Data Analysis 58

Section 2-7.1: Activity Assays 58

Section 2-7.2: Transmission Electron Microscopy 61

Section 2-7.3: COMSOL Simulations 62

Section 2-8: Chapter 2 References 67

Chapter 3 – Synthesizing and Characterizing Enzyme-Gold Nanoparticle Conjugates 69

Section 3-1: Introduction 69

Section 3-2: Results and Discussion 73

Section 3-2.1: Conjugate Design and Preparation 73

Section 3-2.2: Covalent Attachment of Protein to Gold Nanoparticles 78

Section 3-2.3: Characterization of Protein-Gold Nanoparticle Conjugates 81

Section 3-2.4: Protein Concentration Determination of Conjugates 87

Section 3-2.5: Surface Coverage of Conjugates 89

Section 3-3: Chapter 3 Conclusions 94

Section 3-4: Chapter 3 References 96

Chapter 4 – Driving Enzyme Dynamics with Light in Dihydrofolate Reductase-Gold Nanoparticle Conjugates 102

Section 4-1: Introduction 102

Section 4-2: Results and Discussion 106

Section 4-2.1: Experimental Design of Dihydrofolate Reductase Mutants 106

Section 4-2.2: Characterization of Enzyme-Gold Nanoparticle Conjugates 107

Section 4-2.3: Monitoring Activity of DHFR-AuNP Conjugates 111

Section 4-2.4: Laser Heating of the DHFR-AuNP Conjugates 113

Section 4-2.5: Pulsed Laser Heating 116

Section 4-2.6: Continuous Wave Laser Heating 121

Section 4-2.7: Comparing Activity with and without Laser 122

Section 4-3: Chapter 4 Conclusions 124

Section 4-4: Chapter 4 References 125

Chapter 5 – Exploration of Heating Effect 130

Section 5-1: Introduction 130

Section 5-2: Results and Discussion 131

Section 5-2.1: COMSOL Simulations 131

Section 5-2.2: Temperature Dependent Kinetics of DHFR-AuNP Conjugates 135

Section 5-2.3: TEM of Conjugates Before and After Laser Excitation 139

Section 5-3: Chapter 5 Conclusions 143

Section 5-4: Chapter 5 References 146

Chapter 6 – Conclusions 149

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Analytical Chemistry, Biochemistry Chemistry, Physical
Mot-clé	photoexcitation heat flow enzymes catalysis nanoparticles spectroscopy dynamics motions
Committee Chair / Thesis Advisor	R. Brian Dyer, Emory University
Committee Members	Vincent Conticello, Emory University James Kindt, Emory University

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