Regulation and Characterization of Amyloid Architectures: Insight into Amyloid Cytotoxicity and Molecular Self-Assembly Open Access

Ni, Rong (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/gf06g285b?locale=en
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Abstract


Abstract
Regulation and Characterization of Amyloid Architectures:
Insight into Amyloid Cytotoxicity and Molecular
Self-Assembly

by Rong Ni

Amyloid assemblies have been widely studied, leading to identification of the
amyloid-lipid interaction as one the most important factors on regulating amyloid
cytotoxicity. Particularly, the amyloid β-peptides, as the source of naturally occurring
building blocks, have been exploited in the nano-technology field. Unfortunately, the
proposed 3-D structure models of amyloid fibrils and tubes did not provide enough
information on the interactions at the bilayer interface. To unveil some new aspects of
amyloid cytotoxicity and identify the role of bilayer interaction on molecular self-
assembly, the extensive structural characterizations have been performed on the
covalently coupled lipid-Aβ(16-22) hybrid assemblies.
This study led to a discovery of novel Aβ-peptide-amphiphile architectures, which
arise from direct interaction between Aβ-peptide and the lipid alkanes through bending
the terminal alkanes to fit in laminates without disrupting β-sheet structure. The
formation of these architectures is independent on the sample incubation pH, the linking
position of alkane (at either N or C-termini) and peptide orientation/registry,
suggesting an inherent interaction between membrane lipid and amyloid.

During the course of this study, the clear morphology and structure transitions have
been observed when tuning the length or degree of bulkiness of terminal alkanes. Most
strikingly, cross-sheet electrostatic interaction, not the hydrophobic clustering,

has been identified as an important driving force to direct and stabilize the
parallel β-sheet fibril formation.
In addition, we also evaluated the role of glutamine side chain H-bonding cross-
strand pairing on amyloid assembled morphology and peptide organization, shedding
light on the mechanism of glutamine-contained amyloid diseases. In another project, the
peptide strand conformation within the longer amyloid peptide fragment (Aβ(10-35)) has
been roughly defined using fluorescence resonance energy transfer (FRET) technology
with a small FRET pair on four different positions. This conformation provides extra
constraints for study of Aβ(10-35) fibrils structure.
Discoveries in this study are complementary to our current knowledge,
deepening our understanding of amyloid cytotoxicity mechanism and expanding our
regulation factors in the design of novel nanomaterials with
desired morphology and structure.

Table of Contents

Table of Contents
Acknowledgements
List of Figures
List of Tables
Abbreviations

Chapter 1: Amyloid Cytotoxicity and Molecular Self-Assembly Based

Nanomaterials: General Introduction ....................................................... 1

Alzheimer's disease and the amyloid β-peptide assembly................................. 1
Production of amyloid β-peptide ...................................................................... 2
Structure of Aβ fibrils ....................................................................................... 4
Amyloid-lipid interaction.................................................................................. 7
Lipid accelerates Aβ formation......................................................................... 8
Mechanism of amyloid cytotoxicity ............................................................... 10
Molecular self-assembly based nanomaterials ................................................. 14
Biomolecule-based self-assembly................................................................... 14
General forces that govern peptide self-assembly ......................................... 24
Summary and the proposed strategies for pathological and nanotechnogical
studies.......................................................................................................... 29
References............................................................................................................ 32

Chapter 2: Amyloid Peptide / Lipid Chimeras Dictate Parallel β-Sheet Assembly:

the Role of Electrostatics........................................................................... 49
Introduction......................................................................................................... 49
Results
.................................................................................................................. 53
Is the self-assembled morphology altered by addition of alkyl chains at neutral
pH? ................................................................................................................. 53
Does the N-terminal alkyl chain alter the peptide arrangement along the sheets?....... 54
Does the peptide orientation switch at N-propyl? .......................................... 57
What is the peptide arrangement within β-sheets for the N-propyl- to N-
palmityl-Aβ(16-22) fibrils at neutral pH? ...................................................... 58
Does N-terminal alkyl chain interaction direct parallel β-sheet formation? .. 59

Does the cross-sheet electrostatic interaction modulate the peptide
arrangement? .................................................................................................. 61
Do N-terminal alkyl chains alter the distribution of lysine on the fibril surface?............. 67
Does the N-terminal alkyl chain alter the peptide repeat distances? ............. 68
ssNMR characterization of β-sheet stacking .................................................. 70
Does N-isobutyl-Aβ(16-22) L17N-methyl-Q maintain the self-assembly
properties as N-isobutyl-Aβ(16-22)? ............................................................. 72
Characterization of sheets orientation through measuring the 13C-15N cross-
sheet distance with solid-state NMR (ssNMR) REDOR technique................ 73
Discussion............................................................................................................. 74
Materials and methods ....................................................................................... 80
References............................................................................................................ 84

Chapter 3: Accommodating A lkyl C hains within A myloid N anotubes ........................ 87
Introduction......................................................................................................... 87
Results .................................................................................................................. 88
Is the self-assembled morphology at acidic pH altered by addition of alkyl
chains? ............................................................................................................ 88
Do N-terminal alkyl chains impact peptide secondary structure in the
assembly?........................................................................................................ 90
Do the N-alkanes alter the peptide arrangement within β-sheets?.................. 92
Does a N-terminal alkyl chain impact tube wall thickness and tube surface
property? ......................................................................................................... 94
Do the N-terminal alkyl chains alter the peptide repeat distances? ............... 99
What is the conformation of N-lauryl chain within nanotubes? .................. 102
Discussion........................................................................................................... 108
Materials and methods ..................................................................................... 115
References.......................................................................................................... 118

Chapter 4: Exploiting Amyloid Plasticity for Architectural Control I.................... 122
Introduction....................................................................................................... 122
Results ................................................................................................................ 124
Is morphology controlled by the N-terminal cap? ....................................... 124
Impact on assembled secondary structure..................................................... 127
Impact on assembled peptide registry........................................................... 129
Impact on the cross-β amyloid structure....................................................... 131
Structure modeling...................................................................... 132

Discussion........................................................................................................... 136
Materials and methods..................................................................................... 140
References.......................................................................................................... 143


Chapter 5: Exploiting Amyloid Plasticity for Architectural Control II ................. 145
Introduction....................................................................................................... 145
Results................................................................................................................ 146
Is morphology controlled by the C-terminal cap? ....................................... 146
Impact on assembled secondary structure..................................................... 148
Impact on β-sheet laminates ......................................................................... 150
Impact of the C-N(CH3)2 substitution on morphology ............................... 150
Impact of the C-N(CH3)2 substitution on bilayer ........................................ 152
Impact of the C-N(CH3)2 substitution on peptide arrangement .................. 155
Impact of the C-N(CH3)2 substitution on β-sheet laminates ........................ 156
Impact of mixing N- and C-terminal alkyl substitutions on assembled
morphology and peptide arrangement ......................................................... 157
Impact of mixing N- and C-terminal alkyl substitution on β-sheet laminates
.................................................................................................................... 163
Discussion........................................................................................................... 164
Materials and methods..................................................................................... 168
References.......................................................................................................... 171


Chapter 6: Glutamine Cross-Strand Pairing in Amyloid Assembly........................ 173
Introduction....................................................................................................... 173
Results................................................................................................................ 174
Impact of Q substitution on the self-assembly of N-acetyl-Aβ(16-22) ....... 174
The focus on position 22............................................................................... 178
Analysis of the specific E22Q contribution to peptide self-assembly .......... 182
Discussion........................................................................................................... 187
Materials and methods..................................................................................... 193
References.......................................................................................................... 196


Chapter 7: Probing Aβ(10-35) Fibril Structure by Fluorescence Resonance Energy
Transfer (FRET)
..................................................................................... 198
Introduction....................................................................................................... 198
Results................................................................................................................ 200

Selection and placement of FRET pairs........................................................ 200
Assembly of labeled peptides ....................................................................... 205
Fluorescence resonance energy transfer in co-assembled fibrils.................. 211
Discussion........................................................................................................... 216
Materials and methods..................................................................................... 222
References.......................................................................................................... 226


Chapter 8. Conclusions and Outlook .......................................................................... 230
References.......................................................................................................... 236

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