Amyloid Prions and Other Protein Aggregates

II......Page 1
V-VI......Page 2
VII-VIII......Page 4
Preface......Page 6
XIII-XXXVI......Page 9
Introduction......Page 33
Manipulation of the Polyglutamine Tract Length Using Cassette Mutagenesis......Page 35
Expression of Ataxin-3 Variants......Page 39
Purification of His6-Tagged Ataxin-3 Variants......Page 40
Purification of Ataxin-3 Using Fusion Partner Proteins......Page 41
Prevention of Aggregation During Purification......Page 42
Undesirable Proteolysis of the Purified Protein......Page 44
Conformational Analysis of Purified Proteins......Page 45
Functional Analysis of Purified Proteins......Page 46
Conclusion......Page 48
References......Page 49
Background......Page 52
Pre Facto Considerations for Studies of Abeta and Other Amyloid Proteins......Page 53
Metastability and Polydispersity......Page 54
Irreproducibility......Page 55
Introduction......Page 56
The Method......Page 57
Preparation of LMW Abeta by Filtration......Page 58
Preparation of LMW Abeta by Size Exclusion Chromatography......Page 59
Preparation of LMW Abeta by In Situ Filtration......Page 61
References......Page 62
Introduction......Page 66
Disaggregation of Peptides......Page 69
Experimental Results......Page 70
Disaggregation of Polyglutamine Peptides......Page 73
Disaggregation of Abeta(1-40) Peptides......Page 74
Preparation of Stocks of Aggregate Seeds......Page 75
Experimental Results......Page 76
Growth and Storage of Abeta(1-40) Aggregates Under Conditions of Quiescent Growth......Page 78
Growth and Storage of polyGln Aggregates (-20deg)......Page 79
An HPLC Sedimentation Assay for Amyloid Transformations......Page 80
Experimental Results......Page 81
Construction of the Standard Curve......Page 82
Determination of Autosampler Storage Conditions......Page 84
Titration of Fibril Growing Ends......Page 85
Experimental Results......Page 86
Determination of the Growth Site Concentration of an Aggregate Suspension......Page 89
Elongation Kinetics......Page 90
Experimental Results......Page 93
Determination of the Pseudo-First-Order Rate Constant psi1k+......Page 94
Nucleation Kinetics Analysis......Page 95
Experimental Results......Page 97
Nucleation Kinetics Analysis......Page 98
Thermodynamics of Amyloid Fibril Elongation......Page 99
Experimental Results......Page 100
Concluding Remarks......Page 102
References......Page 103
Introduction......Page 107
Identification of Conditions That Promote Aggregation......Page 108
Selection of Conditions in Which the Protein Is Initially in Its Native Conformation......Page 109
Elucidating the Mechanism of Aggregation......Page 111
Selection of the Aggregating Conditions and Assessment of an Initial Native-Like Structure......Page 113
Determination of the Disaggregation Rate Constant (kdis)......Page 114
Determination of the kagg from the Partially Folded State......Page 115
Determination of the kIN and kNI......Page 116
Utilization of the Relevant Rate Constants to Gain Information on the Mechanism of HypF-N Aggregation......Page 117
Assessment of the Native-Like Structure of the Initial Conformation......Page 119
Utilization of the Relevant Rate Constants to Gain Information on the Mechanism of Sso AcP Aggregation......Page 120
Conclusions......Page 121
References......Page 122
Direct Observation of Amyloid Growth Monitored by Total Internal Reflection Fluorescence Microscopy......Page 124
Introduction......Page 125
TIRFM......Page 127
Observation of Fibrils Prepared in Test Tube......Page 128
Real-Time Observation of Fibril Growth......Page 129
Single Fibrillar Analysis of Abeta(1-40) Fibril Growth......Page 131
Conclusion......Page 133
References......Page 134
Introduction......Page 136
Determination of Secondary Structure......Page 137
Determination of Tertiary Structure......Page 140
Determination of Quaternary Structure......Page 146
Construction of Molecular Models Based on Solid State NMR Data......Page 148
Sample Preparation for Solid State NMR......Page 150
References......Page 151
Spin Labeling Analysis of Amyloids and Other Protein Aggregates......Page 156
Introduction......Page 157
Mobility......Page 158
Accessibility......Page 160
Application to Amyloids and Other Protein Aggregates......Page 161
Purification......Page 162
Labeling......Page 163
Sample Holder (Capillaries)......Page 164
Strategy for Distance and Mobility Measurements......Page 165
Accessibility......Page 167
Experimental Considerations......Page 169
Outlook......Page 170
References......Page 171
Introduction......Page 174
Technological Considerations in H/D Exchange-MS......Page 176
Sample Preparation and Buffer Exchange of Monomers......Page 178
Isolation and Buffer Exchange of Amyloid Fibrils......Page 179
Isolation and Buffer Exchange of Protofibrils......Page 180
Dissolution of Aggregates......Page 181
The Artifactual Exchange Problem......Page 182
Minimizing Artifactual Exchange......Page 183
Correction for Artifactual Exchange......Page 184
H/D Exchange into Abeta(1-40) Fibrils......Page 186
Achieving Higher Spatial Resolution by On-Line H/D Exchange-MS with Proteolysis......Page 190
On-Line Proteolysis Using Triaxial Probe......Page 191
[20-34]2+......Page 193
[1-19]5+......Page 195
[35-40]1+......Page 196
References......Page 197
Introduction......Page 201
H/D Exchange, Pepsin Digestion, and Sample Preparation for MALDI-HXMS......Page 204
Data Analysis......Page 205
Notes......Page 206
In- and Back-Exchange......Page 207
Peptide Mapping......Page 208
Results......Page 210
Future Perspective: Improving the Resolution of HXMS Experiments......Page 212
References......Page 213
Introduction......Page 216
Chemically Modified Cysteines to Explore Local Environment Within the Amyloid Fibril......Page 217
Experimental Results......Page 218
Chemical Modification of Sulfhydryl Group of Cys Mutants......Page 219
Alkylation Accessibility of Free Cys Within Amyloid Fibril Structure......Page 220
Experimental Results......Page 221
Solvent Accessibility Studies......Page 222
Disulfide Cross-Linking of Amyloid Fibrils Containing Reduced Double Cys Mutants......Page 223
Experimental Results......Page 225
Oxidative Cross-Linking of Double Cys Mutant Peptide at Fibril Level......Page 227
Experimental Results......Page 228
Oxidative Cross-Linking of Double Cys Mutants at the Monomer Level......Page 230
References......Page 231
Introduction......Page 233
Preparative Centrifugation......Page 234
Theory......Page 236
Experimental Procedures......Page 239
Analytical Ultracentrifugation......Page 240
Theory......Page 241
Experimental Procedures......Page 242
Data Analysis......Page 244
Interpreting Sedimentation Coefficient Distributions......Page 246
References......Page 248
Structural Study of Metastable Amyloidogenic Protein Oligomers by Photo-Induced Cross-Linking of Unmodified Proteins......Page 252
The Role of Protein Oligomers in Amyloidosis......Page 253
Challenges in Biophysical Characterization of Amyloidogenic Protein Oligomers......Page 254
PICUP Photochemistry......Page 255
Optimizing the Experimental System......Page 257
Scope and Limitations of PICUP......Page 259
PICUP as a Tool for Structural Studies......Page 263
Materials......Page 265
Notes......Page 266
References......Page 268
Introduction......Page 272
Pressure-Induced Population of Folding Intermediates of Amyloidogenic Proteins......Page 274
Pressure-Induced Dissolution of Amyloid Fibrils......Page 275
Pressure-Induced Refolding of Active, Native Proteins from Aggregates and Inclusion Bodies......Page 276
Overview of Approach......Page 277
Pressurization and Depressurization......Page 278
High-Pressure Equipment......Page 279
Pressure and Temperature......Page 280
Protein Concentration......Page 281
Pressurization and Depressurization Rates......Page 282
Redox Shuffling Agents......Page 283
Buffer Conditions......Page 284
References......Page 285
Phage Display Screening for Peptides that Inhibit Polyglutamine Aggregation......Page 289
Introduction......Page 290
Library Selection......Page 291
Screening Phage Display Random Peptide Libraries......Page 292
Anti-M13 Antibody Conjugated with Horseradish Peroxidase (Pharmacia)......Page 293
Immobilize Target Protein in High-Binding Microtiter Plates......Page 295
Wash Protocol......Page 297
Elution......Page 298
Freezing Phage......Page 299
Measuring Binding Activity in Pooled Phage by ELISA......Page 300
Isolation and Propagation of Affinity Purified Phage Clones......Page 301
Phage Isolation......Page 302
Confirmation of Binding Activity of Affinity Purified Phage by ELISA......Page 303
Results of Screening for Phage That Binds Proteins Containing Pathological-Length Polyglutamine Domains......Page 304
In Vitro......Page 305
QBP1 Activity in Cells......Page 306
Conclusion......Page 307
References......Page 308
Further Reading......Page 309
Peptide-Based Inhibitors of Amyloid Assembly......Page 310
Rationale for Developing Peptide-Based Fibrillogenesis Inhibitors......Page 311
Random Selection of Sequences......Page 314
Rational Design of Fibrillogenesis Inhibitors......Page 315
Internal Segments of Fibril-Forming Peptides as Fibrillogenesis Inhibitors......Page 316
Amino Acid Substitutions and Side Chain Modifications of Fibrillogenic Domains......Page 317
Proline Substitutions and Additions......Page 318
Charged Blocks Added to Recognition Domains......Page 319
Polyhydroxy and Other Uncharged Polar Blocks......Page 320
N-Terminal Modifications......Page 321
N-Methylation......Page 322
Ester Bonds......Page 330
Modification of alpha-Carbon......Page 331
Peptide Cyclization......Page 332
Use of D-Amino Acids......Page 333
Fibrillogenesis Inhibitors Designed as Nonpeptidic beta-Sheet Mimics......Page 336
Ester Inhibitors......Page 337
Inhibition and Disassembly......Page 338
Production of Fibrils......Page 339
Assays of Fibril Formation......Page 340
Binding of Inhibitor Peptides......Page 341
Application of Inhibitor Peptides to Therapeutic Strategies......Page 343
References......Page 344
Further Reading......Page 349
Introduction......Page 350
Aggregates and Biotinylated Peptides......Page 352
Screening Assay......Page 354
Remarks......Page 356
Results......Page 357
Acknowledgment......Page 360
References......Page 361
Common Pathways of Amyloid Formation......Page 363
An Antibody That Recognizes a Generic Epitope Common to Amyloid Oligomers......Page 365
Preparation of Homogeneous Populations of Stable Oligomers......Page 366
Novel Nanoparticle Molecular Mimic of the Spherical Amyloid Oligomers......Page 368
Activation of the Resin and Displacement by Thiols......Page 369
Preparation and Characterization of the Antigen......Page 370
Antibody Production and Characterization of Conformational Specificity......Page 371
Applications of Oligomer-Specific Conformation-Dependent Antibodies......Page 374
Conclusions......Page 377
References......Page 378
Author Index......Page 382
Subject Index......Page 406