Amyloid, Prions, and Other Protein Aggregates, Part C (eBook)

Cover 1
Copyright page 5
Table of Contents 6
Contributors to Volume 413 8
Preface 10
Volume in series 14
Chapter 1: Purification of Polyglutamine Proteins 38
Abstract 38
Introduction 38
Ataxin-3 Constructs Used 40
Manipulation of the Polyglutamine Tract Length Using Cassette Mutagenesis 40
Expression and Purification of Ataxin-3 44
Quality Control and Analysis of Purified Ataxin-3 47
Conclusion 53
References 54
Chapter 2: Preparation of Amyloid beta-Protein for Structural and Functional Studies 57
Abstract 57
Background 57
Theoretical and Practical Aspects of Amyloid Protein Preparation 58
Preparing Abeta for Biophysical and Biological Study 61
Acknowledgments 67
References 67
Chapter 3: Kinetics and Thermodynamics of Amyloid Assembly Using a High-Performance Liquid Chromatography-Based Sedimentation Assay 71
Abstract 71
Introduction 71
Disaggregation of Peptides 74
Preparation of Stocks of Aggregate Seeds 80
An HPLC Sedimentation Assay for Amyloid Transformations 85
Titration of Fibril Growing Ends 90
Elongation Kinetics 95
Nucleation Kinetics Analysis 100
Thermodynamics of Amyloid Fibril Elongation 104
Concluding Remarks 107
Acknowledgment 108
References 108
Chapter 4: Protein Aggregation Starting From The Native Globular State 112
Abstract 112
Introduction 112
Basic Features of Our Approach 113
Aggregation Mechanism of HypF-N 118
Aggregation Mechanism of Sso AcP 124
Conclusions 126
References 127
Chapter 5: Direct Observation of Amyloid Growth Monitored by Total Internal Reflection Fluorescence Microscopy 128
Abstract 128
Introduction 129
TIRFM 131
Direct Observation of Amyloid beta (1-40) Fibrils 132
Conclusion 137
Acknowledgments 138
References 138
Chapter 6: Characterization of Amyloid Structures at the Molecular Level by Solid State Nuclear Magnetic Resonance Spectroscopy 140
Abstract 140
Introduction 140
Determination of Secondary Structure 141
Determination of Tertiary Structure 144
Determination of Quaternary Structure 150
Construction of Molecular Models Based on Solid State NMR Data 152
Sample Preparation for Solid State NMR 154
Acknowledgments 155
References 155
Chapter 7: Spin Labeling Analysis of Amyloids and Other Protein Aggregates 159
Abstract 159
Introduction 160
Application to Amyloids and Other Protein Aggregates 164
Outlook 173
Acknowledgments 174
References 174
Chapter 8: Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of Protein Aggregates 177
Abstract 177
Introduction 177
Technological Considerations in H/D Exchange-MS 179
H/D Exchange into Abeta(1-40) Fibrils 189
Achieving Higher Spatial Resolution by On-Line H/D Exchange-MS with Proteolysis 193
Proteolysis of Abeta(1-40) Amyloid Fibrils 196
Conclusions 200
Acknowledgments 200
References 200
Chapter 9: Hydrogen-Deuterium Exchange Analyzed by Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry and the HET-s Prion Model 204
Abstract 204
Introduction 204
Materials and Methods 207
Results 213
Future Perspective: Improving the Resolution of HXMS Experiments 215
Conclusion 216
Acknowledgments 216
References 216
Chapter 10: Analysis of Amyloid Fibril Structure by Scanning Cysteine Mutagenesis 219
Abstract 219
Introduction 219
Chemically Modified Cysteines to Explore Local Environment Within the Amyloid Fibril 220
Alkylation Accessibility of Free Cys Within Amyloid Fibril Structure 223
Disulfide Cross-Linking of Amyloid Fibrils Containing Reduced Double Cys Mutants 226
Thermodynamic Stability of Amyloid Fibrils Grown from Cross-Linked Double Cys Mutants 231
Acknowledgments 234
References 234
Chapter 11: Sedimentation Velocity Analysis of Amyloid Oligomers and Fibrils 236
Abstract 236
Introduction 236
Preparative Centrifugation 237
Analytical Ultracentrifugation 243
References 251
Chapter 12: Structural Study of Metastable Amyloidogenic Protein Oligomers by Photo-Induced Cross-Linking of Unmodified Proteins 254
Abstract 254
Introduction 255
PICUP 257
Experimental Protocol 267
Acknowledgments 270
References 270
Chapter 13: High-Pressure Studies on Protein Aggregates and Amyloid Fibrils 274
Abstract 274
Introduction 274
Pressure-Induced Population of Folding Intermediates of Amyloidogenic Proteins 276
Pressure-Induced Aggregation and Amyloid Fibril Formation 277
Pressure-Induced Dissolution of Amyloid Fibrils 277
Pressure-Induced Refolding of Active, Native Proteins from Aggregates and Inclusion Bodies 278
Optimization Parameters for Protein Refolding from Aggregates and Amyloid Fibrils 279
Perspectives 287
Acknowledgment 287
References 287
Chapter 14: Phage Display Screening for Peptides that Inhibit Polyglutamine Aggregation 290
Abstract 290
Introduction 291
Reagents/Solutions 294
Methods 296
Conclusion 308
Acknowledgments 309
References 309
Chapter 15: Peptide-Based Inhibitors of Amyloid Assembly 310
Abstract 310
Rationale for Developing Peptide-Based Fibrillogenesis Inhibitors 311
Approaches to the Design of Peptide-Based Inhibitors 314
Fibrillogenesis Inhibitors Designed as Nonpeptidic beta-Sheet Mimics 336
Methods of Inhibitor Peptide Synthesis and Assessment of Inhibition 337
References 344
Chapter 16: Screening for Modulators of Aggregation with a Microplate Elongation Assay 350
Abstract 350
Introduction 350
Methods 352
Results 357
Acknowledgment 360
References 361
Chapter 17: Conformation-Dependent Anti-Amyloid Oligomer Antibodies 363
Abstract 363
Common Pathways of Amyloid Formation 363
An Antibody That Recognizes a Generic Epitope Common to Amyloid Oligomers 365
Preparation of Homogeneous Populations of Stable Oligomers 366
Novel Nanoparticle Molecular Mimic of the Spherical Amyloid Oligomers 368
Antibody Production and Characterization of Conformational Specificity 371
Applications of Oligomer-Specific Conformation-Dependent Antibodies 374
Conclusions 377
Acknowledgments 378
References 378
Author Index 382
Subject Index 406