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Enzymology at the Membrane Interface: Intramembrane Proteases - 1st Edition - ISBN: 9780128122136, 9780128122143

Enzymology at the Membrane Interface: Intramembrane Proteases, Volume 584

1st Edition

Serial Volume Editor: Michael Gelb
Hardcover ISBN: 9780128122136
eBook ISBN: 9780128122143
Imprint: Academic Press
Published Date: 5th January 2017
Page Count: 494
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Table of Contents

Chapter One: Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease

  • Abstract
  • 1 Introduction
  • 2 The Use of MBP-Tagged Model Substrates for Analysis of Proteolytic Activity of RseP
  • 3 In Vitro Analysis of Proteolysis of SPs by RseP
  • 4 Structural Analysis of RseP Using Thiol-Specific Modification Reagents
  • 5 In Vivo Cross-Linking Analysis to Probe the Interaction of RseP With Its Substrate in the Membrane
  • Acknowledgments

Chapter Two: Signal Peptidase Enzymology and Substrate Specificity Profiling

  • Abstract
  • 1 Introduction
  • 2 Purification of Δ2-75 SP1
  • 3 Purification of SP1 Substrate pONA
  • 4 In Vitro Signal Peptide Cleavage Assay
  • 5 In Vivo Assay of Signal Peptidase Activity
  • 6 Substrate Specificity Profiling
  • 7 Conclusions
  • Acknowledgment

Chapter Three: Screening and Characterization Strategies for Nanobodies Targeting Membrane Proteins

  • Abstract
  • 1 Introduction
  • 2 Phage Display
  • 3 Critical Aspects of Biopannings
  • 4 Screening Strategies
  • 5 Characterization of Nanobodies
  • 6 Potential Applications
  • Acknowledgments

Chapter Four: Probing the Activity of Eukaryotic Rhomboid Proteases In Vitro

  • Abstract
  • 1 Introduction
  • 2 Expression and Solubilization of Rhomboid Proteases From Bacteria
  • 3 In Vitro Probing of Rhomboid Protease Activity
  • Acknowledgments

Chapter Five: Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases

  • Abstract
  • 1 Introduction to Intramembrane Proteases
  • 2 Expression and Purification of GlpG, S2P, and PSH
  • 3 Enzymatic Activity Assays for Intramembrane Proteases
  • 4 Expression and Purification of Human γ-Secretase
  • 5 Enzymatic Activity Assay for Human γ-Secretase
  • 6 Structures of I-CLiPs
  • 7 Concluding Remarks
  • Acknowledgments

Chapter Six: Analyzing Amyloid-β Peptide Modulation Profiles and Binding Sites of γ-Secretase Modulators

  • Abstract
  • 1 Introduction
  • 2 Methods
  • Acknowledgments

Chapter Seven: Probing the Structure and Function Relationships of Presenilin by Substituted-Cysteine Accessibility Method

  • Abstract
  • 1 Introduction
  • 2 Principle of Cysteine-Based Structural Analyses of Presenilin
  • 3 Protocol
  • 4 Summary and Perspectives
  • Acknowledgments

Chapter Eight: A New Method to Determine the Transmembrane Conformation of Substrates in Intramembrane Proteolysis by Deep-UV Resonance Raman Spectroscopy

  • Abstract
  • 1 Introduction
  • 2 In Vitro Intramembrane Proteolysis Assay
  • 3 Intramembrane Proteolysis of the Substrate Monitored by dUVRR Spectroscopy
  • 4 Conclusions
  • Acknowledgment

Chapter Nine: An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane

  • Abstract
  • 1 Introduction
  • 2 Enzymatic Considerations for Kinetic Analysis of Proteolysis in Detergent Micelle Systems
  • 3 A New Strategy: Quantitative Analysis in Proteoliposomes
  • 4 Preparation of Liposomes
  • 5 Preparation of Fluorophore-Labeled Substrate
  • 6 Establishing an Inducible and Fluorogenic Intramembrane Protease Assay
  • 7 Real-Time Kinetic Analysis of Membrane-Immersed Proteolysis
  • 8 Summary
  • Acknowledgments

Chapter Ten: Production of Recombinant Rhomboid Proteases

  • Abstract
  • 1 Introduction
  • 2 Overview
  • 3 Example of an Expression and Purification Protocol—The pBAD System
  • Acknowledgments

Chapter Eleven: Mechanism and Inhibition of Rhomboid Proteases

  • Abstract
  • 1 Introduction
  • 2 Rhomboid Protease Mechanism
  • 3 Substrate Specificity of Rhomboid Proteases
  • 4 Rhomboid Protease Inhibitors
  • 5 Concluding Remarks and Perspectives
  • Acknowledgments

Chapter Twelve: Enzymatic Assays for Studying Intramembrane Proteolysis

  • Abstract
  • 1 Introduction
  • 2 Detergent-Solubilized Assay
  • 3 Proteoliposome Assay
  • 4 Inducible Proteoliposome Assay
  • 5 Conclusions
  • Acknowledgments

Chapter Thirteen: Methods for Structural and Functional Analyses of Intramembrane Prenyltransferases in the UbiA Superfamily

  • Abstract
  • 1 Introduction to the UbiA Superfamily
  • 2 Biological Function and Enzymatic Activity of the Subfamilies
  • 3 Predicting the Functional Role of Intramembrane Prenyltransferases by Sequence Clustering Analysis
  • 4 Analysis of Enzymatic Activity
  • 5 Structural Studies of the UbiA Superfamily
  • Acknowledgments

Chapter Fourteen: Functional Study of the Vitamin K Cycle Enzymes in Live Cells

  • Abstract
  • 1 Introduction
  • 2 Cell-Based Assay for Functional Study of Vitamin K Cycle Enzymes
  • 3 Functional Study of VKOR Using TALENs-Mediated Gene Knockout Reporter Cells
  • 4 Functional Study of GGCX Using CRISPR-Cas9-Mediated Gene Knockout Reporter Cells
  • 5 Concluding Remarks

Chapter Fifteen: Activity Assays for Rhomboid Proteases

  • Abstract
  • 1 Introduction
  • 2 In Vivo Assays
  • 3 In Vitro Assays
  • Acknowledgments


Enzymology at the Membrane Interface: Intramembrane Proteases, Volume 584, the latest release in the Methods in Enzymology series, covers a subset of enzymes that work in the environment of the biological cell membrane. This field, called interfacial enzymology, involves a special series of experimental approaches for the isolation and study of these enzymes.

Key Features

  • Covers a subset of enzymes that work in the environment of the biological cell membrane
  • Offers a series of experimental approaches for the isolation and study of enzymes


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


No. of pages:
© Academic Press 2017
5th January 2017
Academic Press
Hardcover ISBN:
eBook ISBN:

Ratings and Reviews

About the Serial Volume Editor

Michael Gelb

Michael H. Gelb studied chemistry and biochemistry as an undergraduate at the University of California at Davis. His Ph.D. studies with Stephen G. Sligar at Yale University led to a better understanding of the catalytic mechanism of cytochrome P450. As an American Cancer Society Postdoctoral Fellow in the laboratory of the late Robert H. Abeles at Brandeis University, Gelb studied a variety of mechanism-based inactivators of serine proteases and developed fluorinated ketones as tight-binding inhibitors of several classes of proteases. In 1985 Gelb became a faculty member in the Departments of Chemistry and Biochemistry at the University of Washington. Major breakthroughs in the group include the development of methods to properly analyze the action of enzymes on membrane surfaces, the discovery of protein prenylation (farnesylation and geranylgeranylation) in mammalian cells (together with John A. Glomset), the development of Isotope-Coded Affinity Tags (ICAT reagents) for proteomic applications (together with Ruedi Aebersold), and the development of newborn screening for lysosomal storage diseases by mass spectrometry.

Affiliations and Expertise

Dept. of Chemistry, University of Washington, Seattle, USA