Serpin Structure and Evolution

Cover_MIE_501......Page 1
Series Page ii.pdf......Page 2
CopyRight iv......Page 4
Contributors Pages xi-xvi......Page 5
Preface Pages xvii-xviii......Page 11
Methods in Enzymology Series, Pages xix-xlviii......Page 13
Introduction......Page 43
Selection of Strain and Expression Plasmid......Page 44
Growth of Yeast......Page 45
Screening Transformants......Page 46
Large-Scale Growth and Induction......Page 47
Lysis......Page 48
Purification......Page 49
Assessing Serpin Activity and Removing Inactive Forms......Page 51
Production of Polymerogenic Serpins......Page 52
References......Page 54
Abstract......Page 55
Choice of vector and gene construct......Page 56
Identification of serpin genes with mRSL cassette exons......Page 255
Expression conditions to improve soluble expression......Page 58
Purification strategies for isolation of recombinant serpins......Page 59
Recovering overexpressed protein-Soluble versus insoluble purification......Page 60
Image processing in serpin polymer EM-Challenges and progress......Page 477
Do not go back-local elevation dynamics......Page 62
Uniform isotopic labeling......Page 63
Nonnatural amino acid incorporation......Page 64
Preparation of AT from Inclusion Bodies......Page 65
Preparation of Soluble AT......Page 66
Optional purification strategies......Page 68
References......Page 69
Abstract......Page 71
Introduction......Page 72
Dynamics of a canonical serine protease inhibitor, α1-antitrypsin studied by H/D exchange and radiolytic footprinting........Page 373
Isolation of cell nuclei from white blood cells......Page 74
Stable, native, and polymeric states......Page 187
Expression and purification of recombinant MENT......Page 75
Determination of kinetic parameters......Page 76
Intrinsic tryptophan fluorescence to assess conformational change......Page 77
Preparation of dsDNA for EMSAs......Page 78
Electrophoretic mobility shift assays......Page 79
The utility of FCS and TCCD for studying polymerization......Page 409
In silico methods......Page 164
Large-scale MNase digestion and preparation of soluble chromatin......Page 80
Sucrose gradient purification of soluble chromatin......Page 81
Deoxynucleoprotein electrophoresis of reconstituted nucleosomes......Page 82
DNP electrophoresis of nucleosome monomers......Page 83
Isolation and fractionation of chicken blood cells......Page 84
ChIP analysis of MENT association with nuclear DNA in situ......Page 85
References......Page 88
Solving Serpin Crystal Structures......Page 91
Introduction......Page 422
Protein Production and Purification......Page 92
Modifications to Aid Crystallization......Page 93
Monte Carlo simulations......Page 344
Experimental Phasing......Page 94
Molecular Replacement......Page 95
Serpin conformational states......Page 96
Example reaction 3: Measurement of the rate of inhibition of plasmin by α2-antiplasmin......Page 271
Identification of proteolytic cleavage sites......Page 98
Development of Cell Models to Assess the Polymerization of Antitrypsin-Adriana Ordóñez......Page 99
Refinement and Validation......Page 100
References......Page 101
Crystallography of Serpins and Serpin Complexes......Page 105
Labeling Serpins and Proteases with Fluorophores......Page 106
The Michaelis Complex......Page 118
Conformational Control of Serpins-Antithrombin and Heparin......Page 119
Normal mode analysis-studying the energy landscape without simulating it......Page 345
Conformational Change and the Formation of the Latent Conformation......Page 120
Abnormal Conformational Change-The delta-Form......Page 121
High-throughput screening (HTS) and the screen reaction......Page 226
Crystallization of Serpins and Serpin Complexes......Page 123
References......Page 124
Serpins and Allosteric Modulation......Page 131
Methods of Inducing Polymerization......Page 132
Angiotensinogen and Its Interaction with Renin......Page 138
Acknowledgments......Page 142
References......Page 143
Serpin-Glycosaminoglycan Interactions......Page 147
Structure and function of serpins: Unique suicide substrates of proteases......Page 148
Kinetic studies using fluorescence spectroscopy......Page 153
Phage Display Methods......Page 284
Isotopic Labeling and Tryptic Digestion......Page 325
Solid-phase binding......Page 156
Isothermal titration calorimetry......Page 158
NMR......Page 159
X-ray crystallography......Page 160
Activity......Page 163
Antithrombin......Page 169
Construction of the substrate specificity models and prediction of cleavable human serpins by granzyme B......Page 304
Protein C inhibitor......Page 172
References......Page 173
Targeting Serpins in High-Throughput and Structure-Based Drug Design......Page 181
Targeting the s4A Site with Peptides in a Pathogenic Variant of α1-antitrypsin......Page 184
PAGE-based readouts: Nondenaturing and urea-native PAGE......Page 186
Physiologic ligand-binding can affect the antiproteolytic function of serpins......Page 224
Chemical modification......Page 435
Introduction to FCS......Page 401
Site mapping......Page 191
Docking......Page 193
Evaluation of fit......Page 195
In silico approaches targeting the hydrophobic pocket flanking beta-sheet A in α1-antitrypsin......Page 196
A jump forward-steered and targeted dynamics......Page 197
Use of mass spectrometry for drug design in serpins......Page 198
Nano-ESI of noncovalent assemblies: Sample and mass spectrometer considerations......Page 199
Characterization of ligand binding......Page 200
Solution based titration protocol......Page 201
A protocol for spFRET on protease-serpin complexes......Page 414
Mass spectrometry of peptide fragments......Page 202
Crystallographic approaches......Page 203
The target protein crystallizes in known conditions-soaking......Page 204
Using NMR spectroscopy for drug discovery in the serpinopathies......Page 205
NMR spectra of serpins......Page 206
NMR strategies for studying ligand binding to the serpins......Page 207
Mammalian Cell Models and Beyond......Page 208
Conclusion......Page 210
References......Page 211
Development of Inhibitors of Plasminogen Activator Inhibitor-1......Page 219
Introduction......Page 220
Monomers and polymers......Page 221
Biologic functions of the serpin mechanism beyond protease inhibition......Page 222
Selection of target PAI-1......Page 227
The significance of compound concentration......Page 232
The molar ratio of PAI-1 to protease......Page 234
Reporter substrates......Page 236
Two sample HTS......Page 237
Intrinsic fluorescence......Page 441
Work-up of mechanisms of action......Page 240
References......Page 242
Bioinformatic Approaches for the Identification of Serpin Genes with Multiple Reactive Site Loop Coding Exons......Page 251
Introduction......Page 252
Procedure for Identification of Serpin Genes with mRSL Cassette Exons......Page 253
Searching for mRSL serpin genes in the genome of a model organism......Page 257
Proteolytic cleavage and short peptides......Page 259
Improving the odds-replica exchange MD......Page 348
Biopanning procedure......Page 260
References......Page 262
Introduction......Page 265
Initial assessment of the rate of inhibition......Page 268
Discontinuous assay......Page 269
Pushing the Limits-Improving Conformational Sampling......Page 270
Example reaction 1: SI calculation using plasmin-antiplasmin......Page 273
References......Page 275
Predicting Serpin/Protease Interactions......Page 279
Introduction......Page 280
Introduction......Page 283
Application of phage display technology for determining protease substrate specificity......Page 285
Substrate phage library design......Page 286
Substrate phage library generation......Page 288
Screening the substrate phage display library......Page 290
Assessing sublibrary enrichment due to action of the protease......Page 292
Sequence analysis of protease-selected recombinant phage......Page 293
Assessing the quality of the data set generated by substrate phage biopanning......Page 294
A protocol for monitoring neuroserpin polymerization with TCCD (based on the work of Klenerman, Lomas, and collaborator......Page 408
Generating a substrate specificity model for PoPS......Page 296
GROningen MAchine for Chemical Simulations (GROMACS)......Page 298
Proteome-wide search of putative human serpins and identification of the RCL sequences as pseudosubstrates of proteases........Page 299
Inference of serpin inhibitory likelihood for a predicted cleavage site according to its PoPS cleavage score and relative......Page 309
Concluding Remarks and Perspective......Page 311
Acknowledgments......Page 312
Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS): N-Terminal Sequencing of Proteins and Proteolytic Cleavage Sites by Quantitative Mass Spectrometry......Page 317
Introduction......Page 318
Overview of ATOMS......Page 319
A general protocol for labeling proteins with maleimide derivatized fluorophores......Page 396
Limited Proteolytic Processing of the Target Protein by the Test Protease In Vitro......Page 324
Identification of Peptides by Liquid Chromatography-Tandem Mass Spectrometry......Page 328
Analysis of the control experiment and definition of the cutoff for elevated ratio data......Page 329
Identification of outliers, natural N-termini, and basal proteolysis products in the control experiment......Page 331
Discussion: Measuring the Effect of Protease Inhibitors on the Generation of Proteolytic Fragments......Page 333
References......Page 334
Computational Methods for Studying Serpin Conformational Change and Structural Plasticity......Page 337
Introduction......Page 338
Biophysical Techniques to Assess Serpin Polymers Formed In Vivo-James Irving, Ugo Ekeowa, Didier Belorgey, and Imran Haq.........Page 466
MD simulation......Page 342
High-temperature simulations......Page 347
Slow progress forward-umbrella sampling......Page 349
Nondynamical Methods......Page 350
Chemistry at HARvard Molecular Mechanics (CHARMM)......Page 351
Visualization......Page 352
Force Fields......Page 353
Hardware......Page 355
Case Study......Page 356
Outlook......Page 358
References......Page 361
Probing Serpin Conformational Change Using Mass Spectrometry and Related Methods......Page 367
Introduction......Page 368
The effects of a stabilizing cavity-filling mutation on the native state dynamics of α1-antitrypsin......Page 376
Effects of glycosylation on the native state dynamics of human α1-antitrypsin......Page 377
Determination of Thermodynamic Stability Using Hydrogen-Deuterium Exchange Combined with Mass Spectrometry......Page 378
Equilibrium unfolding study of WT α1-AT using the pulse-labeling technique......Page 379
Equilibrium unfolding of G117F mutant studied by HXMS......Page 382
Effects of glycosylation on the equilibrium unfolding of α1-AT observed using HXMS......Page 383
"Functional Unfolding" During the NativeCleaved Transition
......Page 385
Investigating the Polymerization Pathway and Polymer Structure of α1-AT by HXMS and Ion Mobility MS......Page 386
Future Prospects......Page 390
Determining Serpin Conformational Distributions with Single Molecule Fluorescence......Page 393
Introduction......Page 395
Overview of Single Molecule Fluorescence Techniques......Page 397
SMF instrumentation......Page 399
Serpin Polymerization......Page 400
Refolding......Page 436
A protocol for monitoring α1AT polymerization with FCS (based on the work by Gai, Cooperman, and collaborators; Chowdhu......Page 405
Introduction to TCCD......Page 406
Introduction to single pair Förster resonance energy transfer......Page 410
Conformational distributions of trypsin-α1AT complexes......Page 412
Acknowledgments......Page 415
References......Page 416
Serpin Polymerization In Vitro......Page 421
Heat......Page 425
Denaturants......Page 433
Low pH......Page 434
General features and kinetic scheme......Page 437
Loss of monomer......Page 438
Native PAGE......Page 440
Circular dichroism......Page 443
Early studies on the effect of mutations......Page 444
Destabilizing and stabilizing mutations......Page 445
Inhibition by small molecules......Page 446
Inhibition by peptides......Page 447
Reversal by peptides......Page 448
Cleaved polymers......Page 450
Loop-sheet mechanism......Page 452
Biochemical methods for distinguishing between models......Page 453
Limited proteolysis......Page 454
Mutations in RCL......Page 456
Conclusions......Page 457
Acknowledgments......Page 458
References......Page 459
The Serpinopathies: Studying Serpin Polymerization In Vivo......Page 463
Introduction to Serpin Polymers and the Serpinopathies-David Lomas......Page 465
Quantitation, glycosylation, and proteolytic digestion......Page 467
Assessment of Serpin Polymers by Electron Microscopy-Bibek Gooptu......Page 470
Electron microscopy......Page 471
Image collection, particle picking, and data processing......Page 476
Development of mAbs to Aberrant Conformers of α1-Antitrypsin and Neuroserpin-Elena Miranda and Juan Pérez......Page 478
Cell cultures and DNA tranfections......Page 483
Screening of surviving clones......Page 484
Transient transfection of COS-7 cells......Page 485
Stable cell lines overexpressing polymerogenic serpin mutants......Page 486
RT-PCR and XBP1 splicing assay......Page 487
Luciferase assay for ATF6 signaling......Page 488
Luciferase assay for NFkappaB ATF6 signaling......Page 489
Neutrophil preparation......Page 490
Neutrophil functional assays......Page 491
Neutrophil chemotaxis......Page 492
The Use of Transgenic Mice to Assess the Hepatic Consequences of Serpin Polymerization. Jeff Teckman......Page 493
The Use of Transgenic Mice to Assess the Pulmonary Consequences of Serpin Polymerization-Sam Alam and Ravi Mahadeva.........Page 495
Obtaining dermal fibroblasts from patient......Page 499
Derivation and maintenance of human IPS cells......Page 501
Generation of hepatocyte-like cells from hIPSCs......Page 502
Acknowledgments......Page 503
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M......Page 523
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