Amyloid: Merging the Properties of Membranes and Enzymes Open Access

Childers, William Seth (2010)

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Amyloid: Merging the Properties of Membranes and Enzymes
By W. Seth Childers
Since the first observation of amyloid deposits more than 150 years ago, amyloid fibers have
played a critical yet uncertain role in neurodegenerative disease. At the core of this uncertainty are
the cross-β variations proposed to lead to distinct amyloid polymorphs that underpin different
phenotypes. Yet it is unclear how peptide sequence directs polymorphism and functional
differences. Here I have used the critical nucleating core of the Alzheimer's Disease peptide, Aβ(16-
22), as a model to address the molecular origins of amyloid polymorphism and understand core
properties that lead to amyloid's diverse functions. Solid-state NMR and diffraction studies revealed
that simple amino acid changes direct structural polymorphs by modulating both β-strand and β-
sheet pleat registry. Interestingly, these short peptides form hollow nanotubes in which peptides
interact via termini interactions to form a peptide bilayer wall. Like membranes, these amyloid
assemblies have diverse conditionally dependent phases including fibers, nanotubes, oligomers,
twisted and helical ribbons, and lamellar bundles of nanotubes. The cross-β assemblies bind small
molecules (e.g. congo red) and precisely template them into organized molecular arrays providing the
spectroscopic origins of congo red's apple-green birefringence. I further demonstrated that these
lysine-rich amyloid assemblies serve as retro-aldolase catalysts. Taken together, I argue that physical
properties of amyloids can best be seen as merging the long-range order of membranes with the
catalytic features of enzymes and that simple amyloid structures could provide an early prebiotic
catalyst leading to the emergence of complex chemical systems.

Table of Contents

Table of Contents

Chapter 1: Amyloid - An Enigmatic Peptide Assembly 1

Amyloid An Historic Structural Challenge 1

Connecting Morphological Variation with Cross-beta Teriary Structure 5

The Next Structural Challenge: Strain Diversity 7

Molecular Origins of Prion Species Barriers 8

Extension of the Strain Concept to Neurodegenerative Disease 11

Dramatic Impact of Amyloid Co-Assembly in Neurodegenerative Disease 11

Intertwined Nature of Amyloid Dyes and Stain Architecture 13

Mounting Unanswered Questions about a Common Protein Fold 14

Chapter 2: Molecular Origins of Cross-beta Strains 23

Methods 25

Secondary Structural Analysis 35

beta-sheet Registry Determined by Solid-State NMR 37

NMR Distance Measurements to Probe Extended Peptide Backbone 44

Probes of beta-sheet Lamination 46

Cross-Strand Pairing Dictates Peptide Registry 52

Orientation of beta-sheet 57

Discussion 60 Chapter 3: Amyloid Peptides Organized as Bilayer Membranes 71 Methods 73

Development of Microscopy Methods to Visualize Nanotube Walls 81

Probing the Bilayer Structure 82

Probing Inner and Outer Bilayer Surfaces 89

Calculation of Surface-to-Volume Ratio 93

Conclusions 96 Chapter 4: The Phases of Peptide Oligomerization 104 Methods 107 Initial Phase Transition 109 Internal Oligomer Structure 114

Creating Nanotubes and Fiber from Oligomers 121

Impact of Filament Width on Morphology 128

Influence of Salts on Peptide Oligomerization 129

Discussion 131 Chapter 5: Specificity in Amyloid Self-Assembly 139

Preliminary Evaluation of Peptide Mixing 141

The Co-assembly of KLVFFAL and KLVFFAV 142

Probing the Degree of Phase Separation 145

Probing Secondary Structural Differences of Assemblies 149

Testing the effect of Decreasing Side-Chain Length 154

Conclusions 157

Chapter 6: Templating Molecular Arrays in Amyloid's Cross-b Grooves 162

Methods 166

Preliminary Analysis of E22L and E22V Nanotubes 170

CR Binds to E22L Nanotubes that Expose the Termini Surface 172

CR Binding does not alter KLVFFAL's Cross-b Structure 174

CR is Oriented Along the Nanotube Surface 176

CR Docking Model 177 CR Binds at High Density 178

Potential Influence of Biphenyl Conformation Change 182

Discussion 183 Chapter 7: Amyloid as a Catalyst 194 Methods 195 Results 198

Docking of Methodol to KLVFFAL Nanotube Surface 203

Structural Analysis and Catalysis 205 Conclusions 208

Chapter 8: Conclusions - Amyloids as Conformationally Rich Catalytic Membranes 211

Amyloid: Encoding Information Through Conformation 211

Amyloid: Conformational Variation in beta-sheet Strand Registry 211

Amyloid: Conformational Variation in beta-sheet Pleat Registry 213

Amyloid as a Peptide Bilayer Membrane 214

Amyloid as a Catalyst 217

Amyloid: A Platform for the Emergence of Chemical Systems 218

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