Effect of Site-Specific Heating on Enzyme Catalysis Público
Kozlowski, Rachel (Spring 2020)
Published
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
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