Advanced Time-Resolved Fluorescence Microscopy Techniques for the Investigation of Peptide Self-Assembly Público
Anthony, Neil Robert (2013)
Published
Abstract
The ubiquitous cross-beta sheet peptide motif is implicated in numerous neurodegenerative diseases while at the same time offers remarkable potential for constructing isomorphic high-performance bionanomaterials. Despite an emerging understanding of the complex folding landscape of cross-beta structures in determining disease etiology and final structure, we lack knowledge of the critical initial stages of nucleation and growth. In this dissertation, I advance our understanding of these key stages in the cross-beta nucleation and growth pathways using cutting-edge microscopy techniques. In addition, I present a new combined time-resolved fluorescence analysis technique with the potential to advance our current understanding of subtle molecular level interactions that play a pivotal role in peptide self-assembly.
Using the central nucleating core of Alzheimer's Amyloid-beta protein, Abeta(16-22), as a model system, utilizing electron, time-resolved, and non-linear microscopy, I capture the initial and transient nucleation stages of peptide assembly into the cross-beta motif. In addition, I have characterized the nucleation pathway, from monomer to paracrystalline nanotubes in terms of morphology and fluorescence lifetime, corroborating the predicted desolvation process that occurs prior to cross-beta nucleation. Concurrently, I have identified unique heterogeneous cross-beta domains contained within individual nanotube structures, which have potential bionanomaterials applications. Finally, I describe a combined fluorescence theory and analysis technique that dramatically increases the sensitivity of current time-resolved techniques. Together these studies demonstrate the potential for advanced microscopy techniques in the identification and characterization of the cross-beta folding pathway, which will further our understanding of both amyloidogenesis and bionanomaterials.
Table of Contents
1 Introduction and Scope 1
1.1 Microscopy in Scientific Research 2
1.2 Alzheimer's Disease and Amyloid-beta 5
1.3 Fluorescence Lifetime and Rh17-22 8
1.4 Bionanomaterials and Exogenous Factors 9
1.5 Time-Resolved Fluorescence Microscopy 10
1.6 Literature Cited 12
2 Principles of Fluorescence for Quantitative Fluorescence
Microscopy 21
2.1 Introduction 22
2.2 What is Fluorescence? 22
2.2.1 Absorption 25
2.2.2 Molecular Excitation Rates 28
2.2.3 One-Photon Excitation 29
2.2.4 Two-Photon Excitation 33
2.3 Fluorescence and Molecular Relaxation Pathways 38
2.3.1 Internal Conversion 38
2.3.2 Fluorescence Emission 40
2.3.3 Quantum Yield 40
2.3.4 Fluorescence Lifetimes 42
2.3.5 Fluorescence Emission Spectra 45
2.4 Non-Radiative Relaxation Pathways 47
2.4.1 Basics of FRET 48
2.4.2 Intersystem Crossing and Phosphorescence 50
2.5 Photo-Selection and Anisotropy 50
2.6 Measuring Fluorescence in the Microscope 51
2.6.1 Sensitivity of Fluorescence Measurements 52
2.6.2 Fluorescence Signals 55
2.6.3 Observation Volumes and Molecular Brightness 56
2.6.4 Saturation 58
2.6.5 Photobleaching 59
2.7 Summary 60
2.8 References 61
3 Imaging Nucleation, Growth and Heterogeneity in Self-Assembled
Amyloid Phases 71
3.1 Abstract 72
3.2 Introduction 73
3.3 Diversity in Cross-beta Assemblies 74
3.4 Particle Phases 79
3.5 Paracrystallization 81
3.6 Paracrystalline Polymorphism 85
3.7 Functional Energy Transfer 88
3.8 Closing Perspective 89
3.9 Acknowledgements 90
3.10 Literature Cited 90
4 Phase Networks of Cross-beta Peptide Assemblies 96
4.1 Abstract 97
4.2 Introduction 98
4.3 Results and Discussion 100
4.3.1 Accessible Cross-beta Phases. 100
4.3.2 Evaluating Internal Structure of the Particles. 103
4.3.3 The Paracrystalline Phases. 107
4.4 Conclusion 115
4.5 Methods 119
4.5.1 Peptide Synthesis, Purification and Assembly 119
4.5.2 Transmission Electron Microscopy Imaging 120
4.5.3 Circular Dichroism and Melting Temperatures 121
4.5.4 Fluorescence Lifetime Sample Preparation 122
4.5.5 Fluorescence Lifetime Imaging Microscopy 122
4.6 Acknowledgments 123
4.7 References 124
5 Mapping Amyloid-beta(16-22) Nucleation Pathways using
Fluorescence Lifetime Imaging Microscopy 134
5.1 Introduction 135
5.2 Results 138
5.3 Discussion 152
5.4 Conclusions 156
5.5 Methods 157
5.5.1 Peptide Synthesis 157
5.5.2 Fluorescence Lifetime Sample Preparation 157
5.5.3 Fluorescence Lifetime Imaging Microscopy 158
5.5.4 In Situ Sample Loading Video 159
5.6 References 160
6 Possible Pathways to the Molten Globule 166
6.1 Introduction 167
6.2 Results 170
6.3 Discussion 181
6.4 Conclusions 182
6.5 Methods 184
6.5.1 Peptide Synthesis and Sample Preparation 184
6.5.2 Laser Scanning Microscopy 184
6.5.3 Fluorescence Lifetime Imaging Microscopy 185
6.5.4 Phasor Analysis 185
6.5.5 Emission Spectra 186
6.5.6 SHG Measurements 186
6.6 References 187
7 Structural Heterogeneities of Self-Assembled Peptide
Nanomaterials 192
7.1 Abstract 193
7.2 Introduction 194
7.3 Results and Discussion 197
7.3.1 Abeta(16-22) Polymorphism 197
7.3.2 Polymorphism of Phe-Phe Assemblies Directed by Abeta(16-22)
205
7.4 Conclusions 208
7.5 Materials and Methods 209
7.5.1 Peptide Synthesis 209
7.5.2 Amyloid Assembly 209
7.5.3 Diphenylalanine Assembly 210
7.5.4 Optical Microscopy 210
7.5.5 Electron Microscopy 211
7.6 Acknowledgments 212
7.7 References 212
8 Global Analysis of Fluorescence Lifetime and Correlation
Spectroscopy: tauFCS 219
8.1 Abstract 220
8.2 Introduction and Background 220
8.3 Theory 224
8.3.1 Two-photon fluorescence measurements 225
8.3.2 Fluorescence Lifetime and FCS Data 229
8.3.3 Possible Additional Constraints 230
8.4 Methods 231
8.4.1 Experimental 232
8.4.2 Analysis 234
8.4.3 Systematic Error 237
8.4.4 Simulation 238
8.5 Results 238
8.5.1 Model Discrimination and resolution 251
8.6 Discussion 255
8.7 Acknowledgments 259
8.8 References 260
9 Discussion 268
9.1 References 272
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