Energetic Contribution of Amyloid Self-assembly 公开

Liang, Yan (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/2b88qc377?locale=zh
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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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