Cross-ß Folds of Amyloid as a Versatile Self-Propagating Catalyst Open Access

Omosun, Tolulope Olayinka (2016)

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While the proportion of functional folded structures that exist within protein sequence space may be small, most peptides appear to access the cross-β fold. These stacks of β-sheets, initially identified in protein misfolding diseases, form highly ordered soluble assemblies that can access polymorphically diverse phases whose template-directed propagation as ordered supramolecular paracrystalline phases is highly responsive to environmental fluctuations. The minimal nucleating core (LVFF) of the Aβ peptide of Alzheimer's disease constituted within the peptide Ac-KLVFFAL-NH2 assembles at neutral pH into homogeneous nanotubes. Each peptide strand is arranged antiparallel in out-of-register sheets, positioning the N-terminal lysine residue outside the H-bonded β-sheet array creating well-defined surfaces on the faces of the hollow nanotubes composed of rows of binding sites, akin to those in naturally occurring enzymes. The amyloid specific dye Congo red (CR) binds to the peptide nanotubes. The proximity of the binding sites was evaluated through polymerization of 6-amino-2-naphthaldeyde, designed to couple end-to-end through imine condensation along the nanotube surface. This substrate in aqueous solutions of peptide nanotubes (Ac-KLVFFAL-NH2, Ac-HLVFFAL-NH2 and Ac-RLVFFAL-NH2) react to form dimers while the analogous 6-N,N-dimethylamino-2-naphthaldehyde substrate binds the nanotubes without condensation. The catalytic range of the peptide assemblies was further explored with methodol for retro-aldol catalysis. These β-rich assemblies show detectable retro-aldolase activity. Subtle changes in peptide sequence and/or assembly conditions significantly impact final morphology, catalyst efficiency and enantioselectivity. These lysine-rich amyloid assemblies also enantioselectively catalyze aldol condensation of 2-acetonaphthone and 6-methoxy-2-naphthaldehyde. Metal ions such as Cu2+, Zn2+, Ni2+ and Co2+ modulates the assemblies of Ac-Aβ(13-21)H14A and NH2-Aβ(13-21)K16A peptides into fibers and ribbons. The Cu2+-Aβ assemblies are capable of redox activity similar to cupro-enzymes while the Zn2+-Aβ fibers catalyzed retro-aldol cleavage reminiscent of class II aldolase, further extending the catalytic range of these self-propagating Aβ assemblies. These results suggest that simple self-propagating peptide assemblies can produce new enzymes, while also providing a new perspective on the metabolic functions that underlie the fifty odd amyloid diseases. And maybe more importantly, they implicate amyloid as a primitive infective life form, struggling to survive in the nutrient rich eukaryotic cell, acquiring new chemical functions as cellular complexity grows.

Table of Contents

Chapter 1: Introduction

Enzyme Catalysis. 1

Supramolecular Catalysis. 4

Amyloids. 5

Supramolecular Assembly of Simple Aβ Peptides. 6

Cross-strand Pairing in Peptide Assembly. 8

Solvent Accessible Faces in Cross-β Assembly. 10

Ligand Binding in Cross-β Assemblies. 13

Chapter 2: Peptide Nanotubes as Selective Condensation Catalyst

Introduction. 23

Results and Discussion. 23

Secondary Structure Analysis of Ac-KLVFFAL-NH2. 23

Ac-KLVFFAL-NH2 Nanotubes as Templates for Imine Condensation. 28

Optimizing Aβ(16-22)E22L for Catalysis. 39

Imine Condensation with K16 Congeners. 45

Conclusion. 47

Materials and Methods. 48

Chapter 3: Retro-aldol Activity of Aβ(16-22) Congeners: The Binding Site

Introduction. 61

Results and Discussion. 65

Evaluation of the Active Sites in the Nanotubes. 65

Number of Peptides per Binding Site. 68

Retro-aldol Reactivity of Peptide Assemblies. 70

Amyloid Structural Control. 73

Lysine Microenvironment is Critical for Catalysis. 76

Enantioselectivity Studies. 81

Turnover Frequency. 87

Kinetic Analysis of the Retro-aldol Catalysis. 88

Conclusion. 98

Materials and Methods. 99

Chapter 4: Retro-aldol activity of Aβ(16-22) congeners: The Active Site Amine

Introduction. 109

Results and Discussion. 109

Modification of the Position of the Catalytic Amine. 109

Evaluation of the Retro-aldol Activity. 120

Kinetic Evaluation of Catalysis. 126

Manipulating the Hydrophobic Groove. 135

Retro-aldol Activities of L17 and L22 Congeners. 148

Conclusion. 153

Materials and Methods. 154

Chapter 5: Peptide Nanotubes as Aldol Catalyst

Introduction. 160

Results and Discussion. 162

Peptide Nanotubes as Catalyst for Aldol Reaction. 162

Product Rebinding. 174

Enantiospecificity of Aldol reaction. 177

Conclusion. 180

Materials and Methods. 181

Chapter 6: Amyloids as a Rogue Enzyme

Introduction. 184

Results and Discussion. 185

Structural Characterization of Aβ assemblies in the presence of Cu2+. 185

Determination of Redox Potentials of Cu-Aβ complexes. 190

Determination of H2O2 Production by Cu2+-Aβ complexes. 195

Structural Characterization of Aβ assemblies in the presence of Zn2+.199

Structural Characterization of Aβ assemblies in the presence of Co2+ and Ni2+. 203

Retroaldol activity of Zinc Assemblies. 207

Conclusion. 210

Materials and Methods. 213

Chapter 7: Conclusion- Amyloid as a Versatile Self-Propagating Catalyst

Origins of the Biosphere. 225

In the Context of Disease. 226

In the Context of Materials. 226

Future Outlook. 227

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