Energetic Contribution of Amyloid Self-assembly Open Access

Abstract

Controlling pathways that distinguish protein folding from misfolding is critical to viability as highlighted by the nearly 40 protein misfolding diseases. The correlated interactions between amino acid side chains and the backbone have been implicated in protein β-structural assembly and stability, yet the relative contributions have been difficult to evaluate directly. In this dissertation, the central core sequence of the Aβ peptide associated with Alzheimer's disease, Aβ(16-22), was developed as an experimental system for evaluating these interactions. Factors which contribute to the hydrophobic core interactions, such as side chain cross-strand pairing along the β-sheet surface, buried solvent accessible surface area in forming an amyloid structure, and the side chain conformational entropy, were shown to determine the characteristic amyloid cross-β structure. The assembly of the Aβ(16-22) model system indicates that a desolvation step occurs during amyloid nucleation. By direct real-time imaging, peptides that give rise to amyloid assemblies undergo hydrophobic collapse to large micron size aggregates which maintain the properties of molten globules. Further, the amyloid growth is nucleated within these dynamic unstructured aggregates; and once the nuclei are formed, the amyloid assembly grows via monomer addition to the highly ordered amyloid ends. These results provide a direct real-time observation of amyloid assemblies, and suggest that therapeutic intervention should focus on cell type-specific surfaces able to template the nucleation of cytotoxic amyloid. In addition, taking advantage of the amyloid cross-β scaffold, a pigment array has been constructed within a paracrystalline amyloid nanotube and Förster energy transfer along the nanotube surface has been demonstrated to self-assembled acceptor dyes, which promises the potential application of using amyloid structures to generate light harvesting antenna.

Table of Contents

CHAPTER 1 MECHANISM OF AMYLOID SELF-ASSEMBLY AND AMYLOID STRUCTURAL FUNCTIONALITY: GENERAL INTRODUCTION ............................1

Protein Misfolding and Amyloidosis ....................................................................1 Pathways of Amyloid Self-assembly ....................................................................7 Structural Characterization of Amyloid ..............................................................10 Amyloid Nanomaterial Fabrication and Application ..........................................13 Summary ...........................................................................................................15 CHAPTER 2 CROSS-STRAND PAIRING AND AMYLOID MORPHOLOGY..........16 INTRODUCTION ...................................................................................................16 MATERIALS AND METHODS .............................................................................19 Peptide Synthesis and Purification .....................................................................19 Tubes and Fibers Assembly ...............................................................................19 Transmission Electron Microscopy (TEM).........................................................20 Small Angle X-ray Scattering (SAXS) ...............................................................20 Wide Angle X-ray Scattering (WAXS) ..............................................................22 Isotope-Edited Fourier Transform Infrared Spectroscopy (IE-FTIR) ..................22 Molecular Modeling...........................................................................................22 RESULTS ...............................................................................................................23 Assembly Morphology.......................................................................................23 Strand Registry ..................................................................................................33 Molecular Modeling...........................................................................................38 DISCUSSION..........................................................................................................44 CHAPTER 3 PARALLEL AND ANTIPARALLEL β-SHEET IN AMYLOID.............48 INTRODUCTION ...................................................................................................48 MATERIALS AND METHODS .............................................................................49 Peptide Synthesis and Purification......................................................................49

Solid State NMR................................................................................................49 Circular Dichroism Spectroscopy (CD) ..............................................................50 MD Simulation .................................................................................................50 RESULTS ...............................................................................................................50 Morphologies of Aβ(16-22) L17 Congener Self-assemblies ..............................50 β-sheets of Aβ(16-22) L17 Congener Self-assemblies........................................59 Molecular Modeling...........................................................................................68 DISCUSSION..........................................................................................................75 CHAPTER 4 HYDROPHOBIC CORE PACKING IN AMYLOID NANOTUBES ......81 INTRODUCTION ...................................................................................................81 MATERIALS AND METHODS .............................................................................84 Electron Diffraction ...........................................................................................84 RESULTS ...............................................................................................................84 β-sheet Strand Registry of Aβ(16-22) Congener Nanotubes ..............................84 Dimensions of Aβ(16-22) Congener Nanotubes .................................................89 Cross-β Structure of Nanotubes..........................................................................92 Pitch Angles and the Number of Lamination in Nanotubes.................................94 Melting Temperature of Nanotubes ....................................................................99 Molecular Modeling ........................................................................................102 DISCUSSION........................................................................................................107 CHAPTER 5 AMYLOID CO-ASSEMBLY.................................................................112 INTRODUCTION .................................................................................................112 MATERIALS AND METHODS ...........................................................................115 Fluorescent Modifications................................................................................115 Co-assembly of Aβ(16-22) and its Congeners ..................................................115 Two Photon Fluorescence Imaging ..................................................................115 RESULTS .............................................................................................................116 Co-assembly of Aβ(16-22) with Rh16-22 and Rh17-22...................................116 Co-assembly of Aβ(16-22) with Fmoc-labeled Peptide (Fmoc16-22) ..............126 Sequence Dependence of Amyloid Co-assembly..............................................130 Co-assembly of Aβ(16-22) with Congeners, V18I, E22L and E22V.................134 Co-assembly of Aβ(16-22) Congeners, L17I and L17V....................................140 DISCUSSION........................................................................................................142 CHAPTER 6 AMYLOID ASSEMBLY THROUGH AN INTERMOLECULAR MOLTEN GLOBULE.................................................................................................145 INTRODUCTION .................................................................................................145 MATERIALS AND METHODS ...........................................................................146 Aβ(16-22) Assembly System Preparation.........................................................146 Fluorescence Correlation Spectroscopy (FCS)..................................................147 Fluorescence Recovery After Photobleaching (FRAP) .....................................148 Dual Color Fluorescence Imaging of Alexa 633 and Rh17-22 .........................149 RESULTS .............................................................................................................149 Large Aggregate Formation during Aβ(16-22) Assembly ................................149 Peptide Dynamics within the Aggregates .........................................................160 Nanotubes Growth ...........................................................................................164 Dynamics in the Aβ(16-22) Assembly Steady State ........................................169 DISCUSSION........................................................................................................174 CHAPTER 7 POTENTIAL APPLICATION OF AMYLOID NANOTUBES.............177 INTRODUCTION .................................................................................................177 MATERIALS AND METHODS ...........................................................................178 Fluorescence Lifetime Imaging ........................................................................178 Calculation of R0..............................................................................................178 Sulfate Bundling ..............................................................................................179 Cryo-SEM .......................................................................................................179 Two Photon Fluorescence Spectroscopy...........................................................180 PART I Light Harvesting Antenna on an Amyloid Scaffold .............................180 PART II Exciton Coupling and Second Harmonic Generation in Amyloid Nanotube Sulfate Bundles ....................................................................187

CHAPTER 8 CONCLUSION AND PERSPECTIVE.................................................195

REFERENCE.............................................................................................................199

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Keyword	Amyloid Self-assembly
Committee Chair / Thesis Advisor	Lynn, David, Emory University
Committee Members	Lutz, Stefan, Emory University Berland, Keith, Emory University Lian, Tim, Emory University

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