Serpin Structure and Evolution - 1st Edition - ISBN: 9780123859501, 9780123859518

Serpin Structure and Evolution, Volume 501

1st Edition

Serial Volume Editors: James Whisstock Phillip Bird
eBook ISBN: 9780123859518
Hardcover ISBN: 9780123859501
Imprint: Academic Press
Published Date: 16th November 2011
Page Count: 552
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Table of Contents

Series Page

Contributors

Preface

Methods in Enzymology

Intracellular Production of Recombinant Serpins in Yeast

1. Introduction

2. Selection of Strain and Expression Plasmid

3. Growth of Yeast

4. Transformation of Yeast

5. Screening Transformants

6. Large-Scale Growth and Induction

7. Lysis

8. Purification

9. Assessing Serpin Activity and Removing Inactive Forms

10. Production of Polymerogenic Serpins

Production of Recombinant Serpins in Escherichia coli

1. Introduction

2. Experimental Procedures for the Production of Serpins in E. coli

3. Some Common Variations Used for Expression of Other Serpins

4. Preparation of AT from Inclusion Bodies

5. Preparation of Soluble AT

Isolation and Characterization of the Nuclear Serpin MENT

1. Introduction

2. Purification of the MENT Protein

3. Protease Inhibition/Serpin Activity

4. MENT Interaction with DNA and Chromatin In Vitro

5. Analysis of MENT Association with Native Chromatin In Situ

Acknowledgments

Solving Serpin Crystal Structures

1. Introduction

2. Protein Production and Purification

3. Modifications to Aid Crystallization

4. Crystallization

5. Experimental Phasing

6. Molecular Replacement

7. Phase Improvement by Density Modification

8. Refinement and Validation

Crystallography of Serpins and Serpin Complexes

1. Introduction

2. First Glimpses of Serpin Structures

3. The Serpin–Enzyme Complex

4. The Michaelis Complex

5. Conformational Control of Serpins—Antithrombin and Heparin

6. Nonconventional Serpin Complexes

7. Conformational Change and the Formation of the Latent Conformation

8. Abnormal Conformational Change—The δ-Form

9. Serpin Polymers

10. Crystallization of Serpins and Serpin Complexes

11. Conclusions

Serpins as Hormone Carriers

1. Serpins and Allosteric Modulation

2. Hormone Carriage—TBG and CBG

3. Temperature Response: A Protein Thermocouple

4. Angiotensinogen and Its Interaction with Renin

Acknowledgments

Serpin–Glycosaminoglycan Interactions

1. Quantitative Methods

2. Qualitative Methods

3. Animal Models

Acknowledgments

Targeting Serpins in High-Throughput and Structure-Based Drug Design

1. Introduction

2. Targeting the s4A Site with Peptides in a Pathogenic Variant of α1-antitrypsin

3. Computational Approaches

4. In vitro Screening of Small Molecules

5. Mammalian Cell Models and Beyond

6. Conclusion

Development of Inhibitors of Plasminogen Activator Inhibitor-1

1. Introduction

2. Serpins as Drug Targets

3. Development of PAI-1 Inhibitors

4. Concluding Remarks

Bioinformatic Approaches for the Identification of Serpin Genes with Multiple Reactive Site Loop Coding Exons

1. Introduction

2. Procedure for Identification of Serpin Genes with mRSL Cassette Exons

3. Conclusion

Acknowledgment

Methods to Measure the Kinetics of Protease Inhibition by Serpins

1. Introduction

2. Determining the Rate of Protease Inhibition (KA)

3. Efficiency of the Serpin Inhibitory Reaction

Acknowledgments

Predicting Serpin/Protease Interactions

1. Introduction

2. Phage Display Methods

3. Sequence Analysis Methods

4. Concluding Remarks and Perspective

Acknowledgments

Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS)

1. Introduction

2. Overview of ATOMS

3. Control Experiment to Determine the Ratio Cutoff and Identify Natural N-Termini, Basal Proteolytic Products, and Outliers

4. Limited Proteolytic Processing of the Target Protein by the Test Protease In Vitro

5. Isotopic Labeling and Tryptic Digestion

6. Identification of Peptides by Liquid Chromatography-Tandem Mass Spectrometry

7. Mass Spectrometry Data Analysis

8. Discussion: Measuring the Effect of Protease Inhibitors on the Generation of Proteolytic Fragments

Acknowledgments

Computational Methods for Studying Serpin Conformational Change and Structural Plasticity

1. Introduction

2. Local to Global Dynamics Simulations

3. Pushing the Limits—Improving Conformational Sampling

4. I Know Where to Go—Directed Simulations

5. Nondynamical Methods

6. Software

7. Force Fields

8. Hardware

9. Case Study

10. Outlook

Probing Serpin Conformational Change Using Mass Spectrometry and Related Methods

1. Introduction

2. Applications of HXMS

3. Determination of Thermodynamic Stability Using Hydrogen–Deuterium Exchange Combined with Mass Spectrometry

4. “Functional Unfolding” During the Native→Cleaved Transition

5. Investigating the Polymerization Pathway and Polymer Structure of α1-AT by HXMS and Ion Mobility MS

6. Future Prospects

Determining Serpin Conformational Distributions with Single Molecule Fluorescence

Abbreviations

1. Introduction

2. Labeling Serpins and Proteases with Fluorophores

3. Overview of Single Molecule Fluorescence Techniques

4. Serpin Polymerization

5. Conformational Distributions of Protease–Serpin Complexes

6. Conclusions and Future Directions

Acknowledgments

Serpin Polymerization In Vitro

1. Introduction

2. Methods of Inducing Polymerization

3. Kinetics of Polymerization

4. Effect of Mutations and “Drugs” on Polymerization

5. Mechanisms of Polymerization

6. Conclusions

Acknowledgments

The Serpinopathies

1. Introduction to Serpin Polymers and the Serpinopathies—David Lomas

2. Biophysical Techniques to Assess Serpin Polymers Formed In Vivo—James Irving, Ugo Ekeowa, Didier Belorgey, and Imran Haq

3. Assessment of Serpin Polymers by Electron Microscopy—Bibek Gooptu

4. Development of mAbs to Aberrant Conformers of α1-Antitrypsin and Neuroserpin—Elena Miranda and Juan Pérez

5. Development of Cell Models to Assess the Polymerization of Antitrypsin—Adriana Ordóñez

6. Development of Cell Models to Assess the Polymerization of Neuroserpin—Elena Miranda, Juan Perez, and Benoit Roussel

7. Detection of the UPR and the OPR in the Serpinopathies—Lucy Dalton, Sally Thomas, Benoit Roussel, and Stefan Marciniak

8. Characterization of the Interaction Between Serpin Polymers and Neutrophils—Helen Parfrey and Edwin Chilvers

9. The Use of Transgenic Mice to Assess the Hepatic Consequences of Serpin Polymerization. Jeff Teckman

10. The Use of Transgenic Mice to Assess the Pulmonary Consequences of Serpin Polymerization—Sam Alam and Ravi Mahadeva

11. Characterization of Serpin Polymerization Using iPS to Generate Hepatocyte-Like Cell Lines—Tamir Rashid and Ludovic Vallier

Acknowledgments


Description

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.

Key Features

  • This volume in the Methods in Enzymology series comprehensively covers the topic of serpins
  • 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

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists


Details

No. of pages:
552
Language:
English
Copyright:
© Academic Press 2011
Published:
Imprint:
Academic Press
eBook ISBN:
9780123859518
Hardcover ISBN:
9780123859501

About the Serial Volume Editors

James Whisstock Serial Volume Editor

Phillip Bird Serial Volume Editor