Membrane Proteins – Engineering, Purification and Crystallization - 1st Edition - ISBN: 9780128021835, 9780128021934

Membrane Proteins – Engineering, Purification and Crystallization, Volume 557

1st Edition

Serial Volume Editors: Arun K. Shukla
eBook ISBN: 9780128021934
Hardcover ISBN: 9780128021835
Imprint: Academic Press
Published Date: 1st May 2015
Page Count: 644
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Table of Contents

  • Preface
  • Section I: Membrane Protein Engineering, Solubilization and Purification
    • Chapter One: Multicolor Fluorescence-Based Screening Toward Structural Analysis of Multiprotein Membrane Complexes
      • Abstract
      • 1 Introduction
      • 2 Production of Fluorescently Labeled TAP Complexes in P. pastoris
      • 3 Production of Fluorescently Labeled TAP Complexes in Mammalian Cells
      • 4 Multicolor Fluorescence-Based Screening Approaches
      • 5 Summary
      • Acknowledgments
    • Chapter Two: A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs
      • Abstract
      • 1 Introduction
      • 2 Overall Strategy
      • 3 Plasmids, Yeast Strains, and Media
      • 4 Library Construction
      • 5 Expression Screening and Functional Assays
      • 6 Results for the Yeast α-Factor Receptor Ste2p
      • Acknowledgments
    • Chapter Three: Membrane Preparation and Solubilization
      • Abstract
      • 1 Introduction
      • 2 Membrane Preparation
      • 3 Solubilization
    • Chapter Four: Amphipathic Agents for Membrane Protein Study
      • Abstract
      • 1 Introduction
      • 2 MP Stability in Membrane Architecture
      • 3 Conventional Detergents
      • 4 Novel Amphipathic Systems
      • 5 Summary of Detergent Properties
      • 6 Detergent Selection
      • 7 Future Direction
    • Chapter Five: Quantification of Detergent Using Colorimetric Methods in Membrane Protein Crystallography
      • Abstract
      • 1 Detergent in Membrane Protein Crystallography
      • 2 2,6-Dimethylphenol Assay for Sugar-Based Detergents
      • 3 Molybdate Assay for Total Phosphate
      • 4 Assay of Bile Salt with Sulfuric Acid
      • 5 Applications of Detergent Measurement
      • 6 Concluding Remarks
      • Acknowledgments
    • Chapter Six: Solubilization of G Protein-Coupled Receptors: A Convenient Strategy to Explore Lipid–Receptor Interaction
      • Abstract
      • 1 G Protein-Coupled Receptors
      • 2 Membrane Lipids in GPCR Organization and Function
      • 3 Cholesterol: An Important Modulator of GPCR Function
      • 4 Membrane Protein Solubilization: An Essential Step Toward Purification
      • 5 Solubilization as a Strategy to Monitor Lipid–Protein Interactions
      • 6 Conclusions and Future Perspectives
      • Acknowledgments
    • Chapter Seven: Overexpression, Isolation, Purification, and Crystallization of NhaA
      • Abstract
      • 1 Introduction
      • 2 Overexpression
      • 3 Isolation of Membranes from Strain Expressing His-tagged NhaA
      • 4 Affinity Purification of His-tagged NhaA
      • 5 Analysis of Protein Quality
      • 6 Crystallization of His-tagged NhaA
      • 7 Functional Assay
      • 8 Preparation of Solutions
      • Acknowledgments
    • Chapter Eight: Purification, Refolding, and Crystallization of the Outer Membrane Protein OmpG from Escherichia coli
      • Abstract
      • 1 Introduction
      • 2 OmpG Production and Purification
      • 3 Protein Analysis
      • 4 Crystallization of OmpG
      • 5 Summary
    • Chapter Nine: Biophysical Approaches to the Study of LeuT, a Prokaryotic Homolog of Neurotransmitter Sodium Symporters
      • Abstract
      • 1 Introduction
      • 2 LeuT Expression, Purification, Crystallization, and Structure Determination
      • 3 Functional Characterization
      • 4 Transport Mechanism Unveiled from Structure, Function, and Computational Biology
      • 5 Summary
      • Acknowledgments
  • Section II: Generation and Use of Antibody Fragments Against Membrane Proteins
    • Chapter Ten: Generation of Recombinant Antibody Fragments for Membrane Protein Crystallization
      • Abstract
      • 1 Introduction
      • 2 Generation of Antibodies for Structural Characterization of Membrane Proteins
      • Acknowledgments
    • Chapter Eleven: Phage Display Selections for Affinity Reagents to Membrane Proteins in Nanodiscs
      • Abstract
      • 1 Introduction
      • 2 Materials and Equipment
      • 3 Methods
      • 4 Notes
      • Acknowledgments
    • Chapter Twelve: Antibody Fragments for Stabilization and Crystallization of G Protein-Coupled Receptors and Their Signaling Complexes
      • Abstract
      • 1 Introduction
      • 2 The Genesis of Antibody Fragment-Mediated Membrane Protein Crystallization
      • 3 Nanobody Technology—Trapping Active GPCR Conformations
      • 4 Antibody Fragments in Visualizing GPCR Signaling Complexes
      • 5 Conclusion and Future Perspective
      • Acknowledgments
  • Section III: Biophysical Studies of Membrane Proteins
    • Chapter Thirteen: Conformational Analysis of G Protein-Coupled Receptor Signaling by Hydrogen/Deuterium Exchange Mass Spectrometry
      • Abstract
      • 1 Introduction
      • 2 Experimental Procedure
      • 3 Conclusion and Perspectives
      • Acknowledgments
    • Chapter Fourteen: EPR Studies of Gating Mechanisms in Ion Channels
      • Abstract
      • 1 SDSL and EPR Spectroscopy to Study Gating Mechanisms in Ion Channels
      • 2 Voltage-Gated Ion Channels
      • 3 Ligand-Gated Ion Channels
      • 4 Future Directions
      • Acknowledgments
    • Chapter Fifteen: Magic-Angle-Spinning Solid-State NMR of Membrane Proteins
      • Abstract
      • 1 Introduction
      • 2 Production of Recombinant Proteins in Escherichia coli
      • 3 Isolation of Cells and Cellular Membranes
      • 4 Purification and Reconstitution of MPs for ssNMR
      • 5 Dedicated ssNMR Experiments
      • 6 Conclusions
      • Acknowledgments
    • Chapter Sixteen: Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins: A Guide to Spin Label Paramagnetic Relaxation Enhancement Restraints
      • Abstract
      • 1 Site-Directed Spin Labeling
      • 2 PRE Measurements
      • 3 Structure Calculation with PRE Restraints
      • 4 Assessment of Structure Quality
      • 5 Future Developments
      • 6 Summary
  • Section IV: Crystallization of Membrane Proteins
    • Chapter Seventeen: Inducing Two-Dimensional Crystallization of Membrane Proteins by Dialysis for Electron Crystallography
      • Abstract
      • 1 Introduction
      • 2 2D Crystallization by Dialysis
      • 3 EM Screening of 2D Crystallization Conditions
      • 4 Optimization of 2D Crystallization and Protein Purification
      • 5 Conclusion
    • Chapter Eighteen: Crystallization of Membrane Proteins by Vapor Diffusion
      • Abstract
      • 1 Introduction
      • 2 Membrane Protein Expression
      • 3 Membrane Protein Purification
      • 4 Membrane Protein Crystallization via Vapor Diffusion
      • 5 Case Studies
      • 6 Concluding Remarks
      • Acknowledgment
    • Chapter Nineteen: Bicelles Coming of Age: An Empirical Approach to Bicelle Crystallization
      • Abstract
      • 1 Introduction
      • 2 Protein Function and Folding in Lipid Bicelles
      • 3 Bicelle Crystallization of MPs
      • 4 User Guide to Bicelle Crystallization
      • 5 Bicelle Crystallization General Protocol
      • 6 Successful Crystallization Conditions
      • 7 Case Study of Cellulose Synthase Crystallization
      • 8 Alternative Approaches
      • 9 Summary
      • Acknowledgments
    • Chapter Twenty: Fluorescence Recovery After Photobleaching in Lipidic Cubic Phase (LCP-FRAP): A Precrystallization Assay for Membrane Proteins
      • Abstract
      • 1 Introduction
      • 2 Experimental Components and Considerations for LCP-FRAP Assays
      • 3 Examples of Using LCP-FRAP to Guide GPCR Crystallization
      • 4 Protocols for LCP-FRAP Assays
      • Acknowledgments
    • Chapter Twenty-One: Crystallization of Proteins from Crude Bovine Rod Outer Segments
      • Abstract
      • 1 Introduction
      • 2 Experimental Procedures
      • 3 Pilot Experimental Results
      • 4 Conclusions
      • Acknowledgments
    • Chapter Twenty-Two: Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser
      • Abstract
      • 1 Introduction
      • 2 Isolation of Photosystem II
      • 3 Crystallization for Studies with FELs
      • 4 Detection and Characterization of Nano- and Microcrystals
      • 5 Time-Resolved Crystallography of PSII Using FELs
      • 6 Summary
      • Acknowledgments
  • Section V: Computational Approaches to Understand Membrane Proteins
    • Chapter Twenty-Three: Major Intrinsic Protein Superfamily: Channels with Unique Structural Features and Diverse Selectivity Filters
      • Abstract
      • 1 Introduction
      • 2 Structural Biology of MIP Channels
      • 3 MIPModDB Database: A Database of MIP Models
      • 4 Unique Structural Features Within the TM Region
      • 5 Residues Forming Ar/R SF Are Diverse
      • 6 Conclusion
      • Acknowledgments
    • Chapter Twenty-Four: Comparative Sequence–Function Analysis of the Major Facilitator Superfamily: The “Mix-and-Match” Method
      • Abstract
      • 1 Introduction
      • 2 Helix-Triplets
      • 3 Detection of Functionally Homologous Positions
      • 4 Examples
      • 5 Conclusion
      • Acknowledgments
    • Chapter Twenty-Five: Elucidating Ligand-Modulated Conformational Landscape of GPCRs Using Cloud-Computing Approaches
      • Abstract
      • 1 Introduction
      • 2 What Are Markov State Models?
      • 3 How Do MSMs Enable Novel Insight?
      • 4 How Can MSMs Be Used for Improved Drug Design?
      • 5 Conclusions
      • Acknowledgments
  • Author Index
  • Subject Index

Description

Membrane Proteins – Engineering, Purification and Crystallization, a volume of Methods In Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol for the interested audience. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines.

Key Features

  • Volume of Methods In Enzymology
  • Contains a collection of a diverse array of topics in the area of membrane protein biology ranging from recombinant expression, isolation, functional characterization, biophysical studies and crystallization

Readership

These two volumes of Methods In Enzymology should be very useful to any researcher working in the area of structure and function of membrane proteins. Graduate students, post-doctoral fellows and faculty members pursuing different aspects of membrane protein biology should find these two volumes useful and complete collection of practical information.


Details

No. of pages:
644
Language:
English
Copyright:
© Academic Press 2015
Published:
Imprint:
Academic Press
eBook ISBN:
9780128021934
Hardcover ISBN:
9780128021835

About the Serial Volume Editors

Arun K. Shukla Serial Volume Editor

Dr. Arun K. Shukla obtained his M.Sc. (Master in Science) from the Center for Biotechnology at the Jawaharlal Nehru University in New Delhi, India. Dr. Shukla did his Ph.D. from the Department of Molecular Membrane Biology at the Max Planck Institute of Biophysics in Frankfurt, Germany. His Ph.D. research work was focused on structural studies of G Protein-Coupled Receptors (GPCRs).

Dr. Shukla subsequently carried out his post-doctoral work in the Department of Medicine at the Duke University in North Carolina, USA. During his post-doctoral research work, Dr. Shukla focused on understanding the biophysical and structural basis of ß-arrestin mediated regulation of GPCRs and non-canonical GPCR signaling. Dr. Shukla has served as an Assistant Professor in the Department of Medicine at the Duke University in Durham, North Carolina, USA.

Dr. Shukla is currently an Assistant Professor in Department of Biological Sciences and Bioengineering at the Indian Institute of Technology, Kanpur, India. Dr. Shukla is also an Intermediate Fellow of the Wellcome Trust-DBT India Alliance. The research program in Dr. Shukla’s laboratory is focused on understanding the molecular mechanism of activation, signaling and regulation of G Protein-Coupled Receptors.

Affiliations and Expertise

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India