Conformational evolution in amyloid self-assembly Público
Liang, Chen (2017)
Published
Abstract
Amyloid has been correlated with numerous protein misfolding diseases, yet no effective treatment is currently available. A thorough understanding of the misfolding process is crucial for understanding the disease mechanism and designing therapeutics. This dissertation presents a conformational evolution model that builds on earlier nucleation dependent assembly and polymorphism models. The nucleating core of Aβ Dutch mutant, Aβ (16-22) E22Q is quantitatively characterized to go through a conformational transition from anti-parallel out-of-register to parallel strand orientation. Solid-state NMR and isotope-edited infrared (IE-IR) spectroscopies are developed to assign basis sets for monitoring and resolving the evolving conformations. The impact of single amino acid mutation and ionic strength of the environment are used to reveal the sensitivity of the self-assembly process to intrinsic and extrinsic modulators along the evolving energy surface.
Significant extension of local and normal IR active modes are uniquely revealed in the extended amyloid secondary structure and these are extended and optimized for the analyses. Overall, the results extend and enhance our current understanding of amyloid assembly for the potential design of therapeutics for neurodegenerative diseases.
Table of Contents
Abstract
Acknowlegements. 6
List of Figures. 13
List of Tables. 21
Chapter 1. Background. 1
Protein self-assembly. 1
Cross-beta structure. 2
Decoding amyloid self-assembly. 4
The self-assembly process. 8
Nucleation dependent self-assembly. 8
Polymorphism in amyloid self-assembly. 10
Conformational evolution in amyloid self-assembly process. 11
References. 22
Chapter 2. Defining conformational distribution of cross-β peptide assembly using isotope edited infrared spectroscopy. 30
Background. 30
Isotope edited infrared spectroscopy in the study of amyloid self-assembly. 31
Methods. 33
Results. 35
Experimental data of basis sets of various β-sheet conformations. 35
The gap at the early stage of assembly. 39
Defining the early time species. 40
Completion of the basis sets. 41
Conclusion. 42
References. 43
Chapter 3. Mapping the kinetic intermediates along amyloid self-assembly. 46
Introduction. 46
Methods. 49
Peptide synthesis and purification: 49
Transmission Electron Microscopy (TEM): 50
Circular Dichroism spectroscopy: 51
Attenuated Total Reflectance Fourier Transform Infrared (AT-FTIR): 51
Results. 52
E22Q assembles through an anti-parallel to parallel β-sheet transition. 52
Measuring the kinetics of conformational transition. 60
Mapping conformational changes with IE-IR fitting. 62
Glutamine side chain is the determining factor of the structural transition. 63
The impact of pathway barrier adjustment on amyloid conformational transition. 65
Seeding experiment supports the lack of parallel nuclei at the beginning of assembly 68
Parallel nuclei is likely to emerge from mutation. 70
Conclusion. 73
Reference. 74
Chapter 4. Ionic effect on the conformational transition of amyloid. 78
Introduction. 78
Methods. 83
Fibril Assembly in the presence of salt 83
Attenuated Total Reflectance Fourier Transform Infrared (AT-FTIR) 83
Fitting of Isotope-Edited FTIR Spectra. 83
Solid state NMR.. 84
Results. 86
Particle phase. 86
Elongation and conversion. 88
Discussion. 96
Conclusion. 99
References. 99
Chapter 5 Positional dependence of Q on amyloid self-assembly. 104
Introduction. 104
Methods. 105
Results. 106
Morphology and transition kinetics of different mutants. 106
Mutation at the nucleating core of Aβ(16-22): F19Q and F20Q.. 112
Pattern analysis. 118
Conclusion. 120
References. 121
Chapter 6 IE-IR of parallel peptide system.. 124
Background. 124
Methods. 127
Results. 128
Scheme design. 128
IE-IR spectra of isotope enrichment at different positions. 130
Dilution experiment 138
Conclusion. 143
References. 144
Chapter 7. Conclusion and perspectives 147
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