Serpins are a group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases. This volume in the Methods in Enzymology series comprehensively covers this topic. With an international board of authors, this volume covers subjects such as Crystallography of serpins and serpin complexes, Serpins as hormone transporters, and Production of serpins using cell free systems.
1. 1. Introduction 2. 2. Selection of Strain and Expression Plasmid 3. 3. Growth of Yeast 4. 4. Transformation of Yeast 5. 5. Screening Transformants 6. 6. Large-Scale Growth and Induction 7. 7. Lysis 8. 8. Purification 9. 9. Assessing Serpin Activity and Removing Inactive Forms 10. 10. Production of Polymerogenic Serpins 11. Production of Recombinant Serpins in Escherichia coli 12. 1. Introduction 13. 2. Experimental Procedures for the Production of Serpins in E. coli 14. 3. Some Common Variations Used for Expression of Other Serpins 15. 4. Preparation of AT from Inclusion Bodies 16. 5. Preparation of Soluble AT 17. Isolation and Characterization of the Nuclear Serpin MENT 18. 1. Introduction 19. 2. Purification of the MENT Protein 20. 3. Protease Inhibition/Serpin Activity 21. 4. MENT Interaction with DNA and Chromatin In Vitro 22. 5. Analysis of MENT Association with Native Chromatin In Situ 23. Acknowledgments 24. Solving Serpin Crystal Structures 25. 1. Introduction 26. 2. Protein Production and Purification 27. 3. Modifications to Aid Crystallization 28. 4. Crystallization 29. 5. Experimental Phasing 30. 6. Molecular Replacement 31. 7. Phase Improvement by Density Modification 32. 8. Refinement and Validation 33. Crystallography of Serpins and Serpin Complexes 34. 1. Introduction 35. 2. First Glimpses of Serpin Structures 36. 3. The Serpin–Enzyme Complex 37. 4. The Michaelis Complex 38. 5. Conformational Control of Serpins—Antithrombin and Heparin 39. 6. Nonconventional Serpin Complexes 40. 7. Conformational Change and the Formation of the Latent Conformation 41. 8. Abnormal Conformational Change—The δ-Form 42. 9. Serpin Polymers 43. 10. Crystallization of Serpins and Serpin Complexes 44. 11. Conclusions 45. Serpins as Hormone Carriers 46. 1. Serpins and Allosteric Modulation 47. 2. Hormone Carriage—TBG and CBG 48. 3. Temperature Response: A Protein Thermocouple 49. 4. Angiotensinogen and Its Interaction with Renin 50. Acknowledgments 51. Serpin–Glycosaminoglycan Interactions 52. 1. Quantitative Methods 53. 2. Qualitative Methods 54. 3. Animal Models 55. Acknowledgments 56. Targeting Serpins in High-Throughput and Structure-Based Drug Design 57. 1. Introduction 58. 2. Targeting the s4A Site with Peptides in a Pathogenic Variant of α1-antitrypsin 59. 3. Computational Approaches 60. 4. In vitro Screening of Small Molecules 61. 5. Mammalian Cell Models and Beyond 62. 6. Conclusion 63. Development of Inhibitors of Plasminogen Activator Inhibitor-1 64. 1. Introduction 65. 2. Serpins as Drug Targets 66. 3. Development of PAI-1 Inhibitors 67. 4. Concluding Remarks 68. Bioinformatic Approaches for the Identification of Serpin Genes with Multiple Reactive Site Loop Coding Exons 69. 1. Introduction 70. 2. Procedure for Identification of Serpin Genes with mRSL Cassette Exons 71. 3. Conclusion 72. Acknowledgment 73. Methods to Measure the Kinetics of Protease Inhibition by Serpins 74. 1. Introduction 75. 2. Determining the Rate of Protease Inhibition (KA) 76. 3. Efficiency of the Serpin Inhibitory Reaction 77. Acknowledgments 78. Predicting Serpin/Protease Interactions 79. 1. Introduction 80. 2. Phage Display Methods 81. 3. Sequence Analysis Methods 82. 4. Concluding Remarks and Perspective 83. Acknowledgments 84. Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) 85. 1. Introduction 86. 2. Overview of ATOMS 87. 3. Control Experiment to Determine the Ratio Cutoff and Identify Natural N-Termini, Basal Proteolytic Products, and Outliers 88. 4. Limited Proteolytic Processing of the Target Protein by the Test Protease In Vitro 89. 5. Isotopic Labeling and Tryptic Digestion 90. 6. Identification of Peptides by Liquid Chromatography-Tandem Mass Spectrometry 91. 7. Mass Spectrometry Data Analysis 92. 8. Discussion: Measuring the Effect of Protease Inhibitors on the Generation of Proteolytic Fragments 93. Acknowledgments 94. Computational Methods for Studying Serpin Conformational Change and Structural Plasticity 95. 1. Introduction 96. 2. Local to Global Dynamics Simulations 97. 3. Pushing the Limits—Improving Conformational Sampling 98. 4. I Know Where to Go—Directed Simulations 99. 5. Nondynamical Methods 100. 6. Software 101. 7. Force Fields 102. 8. Hardware 103. 9. Case Study 104. 10. Outlook 105. Probing Serpin Conformational Change Using Mass Spectrometry and Related Methods 106. 1. Introduction 107. 2. Applications of HXMS 108. 3. Determination of Thermodynamic Stability Using Hydrogen–Deuterium Exchange Combined with Mass Spectrometry 109. 4. “Functional Unfolding” During the Native→Cleaved Transition 110. 5. Investigating the Polymerization Pathway and Polymer Structure of α1-AT by HXMS and Ion Mobility MS 111. 6. Future Prospects 112. Determining Serpin Conformational Distributions with Single Molecule Fluorescence 113. Abbreviations 114. 1. Introduction 115. 2. Labeling Serpins and Proteases with Fluorophores 116. 3. Overview of Single Molecule Fluorescence Techniques 117. 4. Serpin Polymerization 118. 5. Conformational Distributions of Protease–Serpin Complexes 119. 6. Conclusions and Future Directions 120. Acknowledgments 121. Serpin Polymerization In Vitro 122. 1. Introduction 123. 2. Methods of Inducing Polymerization 124. 3. Kinetics of Polymerization 125. 4. Effect of Mutations and “Drugs” on Polymerization 126. 5. Mechanisms of Polymerization 127. 6. Conclusions 128. Acknowledgments 129. The Serpinopathies 130. 1. Introduction to Serpin Polymers and the Serpinopathies—David Lomas 131. 2. Biophysical Techniques to Assess Serpin Polymers Formed In Vivo—James Irving, Ugo Ekeowa, Didier Belorgey, and Imran Haq 132. 3. Assessment of Serpin Polymers by Electron Microscopy—Bibek Gooptu 133. 4. Development of mAbs to Aberrant Conformers of α1-Antitrypsin and Neuroserpin—Elena Miranda and Juan Pérez 134. 5. Development of Cell Models to Assess the Polymerization of Antitrypsin—Adriana Ordóñez 135. 6. Development of Cell Models to Assess the Polymerization of Neuroserpin—Elena Miranda, Juan Perez, and Benoit Roussel 136. 7. Detection of the UPR and the OPR in the Serpinopathies—Lucy Dalton, Sally Thomas, Benoit Roussel, and Stefan Marciniak 137. 8. Characterization of the Interaction Between Serpin Polymers and Neutrophils—Helen Parfrey and Edwin Chilvers 138. 9. The Use of Transgenic Mice to Assess the Hepatic Consequences of Serpin Polymerization. Jeff Teckman 139. 10. The Use of Transgenic Mice to Assess the Pulmonary Consequences of Serpin Polymerization—Sam Alam and Ravi Mahadeva 140. 11. Characterization of Serpin Polymerization Using iPS to Generate Hepatocyte-Like Cell Lines—Tamir Rashid and Ludovic Vallier 141. Acknowledgments
Serpins are a group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases. This volume in the Methods in Enzymology series comprehensively covers this topic. With an international board of authors, this volume covers subjects such as Crystallography of serpins and serpin complexes, Serpins as hormone transporters, and Production of serpins using cell free systems.
1. 1. Introduction 2. 2. Selection of Strain and Expression Plasmid 3. 3. Growth of Yeast 4. 4. Transformation of Yeast 5. 5. Screening Transformants 6. 6. Large-Scale Growth and Induction 7. 7. Lysis 8. 8. Purification 9. 9. Assessing Serpin Activity and Removing Inactive Forms 10. 10. Production of Polymerogenic Serpins 11. Production of Recombinant Serpins in Escherichia coli 12. 1. Introduction 13. 2. Experimental Procedures for the Production of Serpins in E. coli 14. 3. Some Common Variations Used for Expression of Other Serpins 15. 4. Preparation of AT from Inclusion Bodies 16. 5. Preparation of Soluble AT 17. Isolation and Characterization of the Nuclear Serpin MENT 18. 1. Introduction 19. 2. Purification of the MENT Protein 20. 3. Protease Inhibition/Serpin Activity 21. 4. MENT Interaction with DNA and Chromatin In Vitro 22. 5. Analysis of MENT Association with Native Chromatin In Situ 23. Acknowledgments 24. Solving Serpin Crystal Structures 25. 1. Introduction 26. 2. Protein Production and Purification 27. 3. Modifications to Aid Crystallization 28. 4. Crystallization 29. 5. Experimental Phasing 30. 6. Molecular Replacement 31. 7. Phase Improvement by Density Modification 32. 8. Refinement and Validation 33. Crystallography of Serpins and Serpin Complexes 34. 1. Introduction 35. 2. First Glimpses of Serpin Structures 36. 3. The Serpin–Enzyme Complex 37. 4. The Michaelis Complex 38. 5. Conformational Control of Serpins—Antithrombin and Heparin 39. 6. Nonconventional Serpin Complexes 40. 7. Conformational Change and the Formation of the Latent Conformation 41. 8. Abnormal Conformational Change—The δ-Form 42. 9. Serpin Polymers 43. 10. Crystallization of Serpins and Serpin Complexes 44. 11. Conclusions 45. Serpins as Hormone Carriers 46. 1. Serpins and Allosteric Modulation 47. 2. Hormone Carriage—TBG and CBG 48. 3. Temperature Response: A Protein Thermocouple 49. 4. Angiotensinogen and Its Interaction with Renin 50. Acknowledgments 51. Serpin–Glycosaminoglycan Interactions 52. 1. Quantitative Methods 53. 2. Qualitative Methods 54. 3. Animal Models 55. Acknowledgments 56. Targeting Serpins in High-Throughput and Structure-Based Drug Design 57. 1. Introduction 58. 2. Targeting the s4A Site with Peptides in a Pathogenic Variant of α1-antitrypsin 59. 3. Computational Approaches 60. 4. In vitro Screening of Small Molecules 61. 5. Mammalian Cell Models and Beyond 62. 6. Conclusion 63. Development of Inhibitors of Plasminogen Activator Inhibitor-1 64. 1. Introduction 65. 2. Serpins as Drug Targets 66. 3. Development of PAI-1 Inhibitors 67. 4. Concluding Remarks 68. Bioinformatic Approaches for the Identification of Serpin Genes with Multiple Reactive Site Loop Coding Exons 69. 1. Introduction 70. 2. Procedure for Identification of Serpin Genes with mRSL Cassette Exons 71. 3. Conclusion 72. Acknowledgment 73. Methods to Measure the Kinetics of Protease Inhibition by Serpins 74. 1. Introduction 75. 2. Determining the Rate of Protease Inhibition (KA) 76. 3. Efficiency of the Serpin Inhibitory Reaction 77. Acknowledgments 78. Predicting Serpin/Protease Interactions 79. 1. Introduction 80. 2. Phage Display Methods 81. 3. Sequence Analysis Methods 82. 4. Concluding Remarks and Perspective 83. Acknowledgments 84. Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) 85. 1. Introduction 86. 2. Overview of ATOMS 87. 3. Control Experiment to Determine the Ratio Cutoff and Identify Natural N-Termini, Basal Proteolytic Products, and Outliers 88. 4. Limited Proteolytic Processing of the Target Protein by the Test Protease In Vitro 89. 5. Isotopic Labeling and Tryptic Digestion 90. 6. Identification of Peptides by Liquid Chromatography-Tandem Mass Spectrometry 91. 7. Mass Spectrometry Data Analysis 92. 8. Discussion: Measuring the Effect of Protease Inhibitors on the Generation of Proteolytic Fragments 93. Acknowledgments 94. Computational Methods for Studying Serpin Conformational Change and Structural Plasticity 95. 1. Introduction 96. 2. Local to Global Dynamics Simulations 97. 3. Pushing the Limits—Improving Conformational Sampling 98. 4. I Know Where to Go—Directed Simulations 99. 5. Nondynamical Methods 100. 6. Software 101. 7. Force Fields 102. 8. Hardware 103. 9. Case Study 104. 10. Outlook 105. Probing Serpin Conformational Change Using Mass Spectrometry and Related Methods 106. 1. Introduction 107. 2. Applications of HXMS 108. 3. Determination of Thermodynamic Stability Using Hydrogen–Deuterium Exchange Combined with Mass Spectrometry 109. 4. “Functional Unfolding” During the Native→Cleaved Transition 110. 5. Investigating the Polymerization Pathway and Polymer Structure of α1-AT by HXMS and Ion Mobility MS 111. 6. Future Prospects 112. Determining Serpin Conformational Distributions with Single Molecule Fluorescence 113. Abbreviations 114. 1. Introduction 115. 2. Labeling Serpins and Proteases with Fluorophores 116. 3. Overview of Single Molecule Fluorescence Techniques 117. 4. Serpin Polymerization 118. 5. Conformational Distributions of Protease–Serpin Complexes 119. 6. Conclusions and Future Directions 120. Acknowledgments 121. Serpin Polymerization In Vitro 122. 1. Introduction 123. 2. Methods of Inducing Polymerization 124. 3. Kinetics of Polymerization 125. 4. Effect of Mutations and “Drugs” on Polymerization 126. 5. Mechanisms of Polymerization 127. 6. Conclusions 128. Acknowledgments 129. The Serpinopathies 130. 1. Introduction to Serpin Polymers and the Serpinopathies—David Lomas 131. 2. Biophysical Techniques to Assess Serpin Polymers Formed In Vivo—James Irving, Ugo Ekeowa, Didier Belorgey, and Imran Haq 132. 3. Assessment of Serpin Polymers by Electron Microscopy—Bibek Gooptu 133. 4. Development of mAbs to Aberrant Conformers of α1-Antitrypsin and Neuroserpin—Elena Miranda and Juan Pérez 134. 5. Development of Cell Models to Assess the Polymerization of Antitrypsin—Adriana Ordóñez 135. 6. Development of Cell Models to Assess the Polymerization of Neuroserpin—Elena Miranda, Juan Perez, and Benoit Roussel 136. 7. Detection of the UPR and the OPR in the Serpinopathies—Lucy Dalton, Sally Thomas, Benoit Roussel, and Stefan Marciniak 137. 8. Characterization of the Interaction Between Serpin Polymers and Neutrophils—Helen Parfrey and Edwin Chilvers 138. 9. The Use of Transgenic Mice to Assess the Hepatic Consequences of Serpin Polymerization. Jeff Teckman 139. 10. The Use of Transgenic Mice to Assess the Pulmonary Consequences of Serpin Polymerization—Sam Alam and Ravi Mahadeva 140. 11. Characterization of Serpin Polymerization Using iPS to Generate Hepatocyte-Like Cell Lines—Tamir Rashid and Ludovic Vallier 141. Acknowledgments
