Enzymology at the Membrane Interface: Intramembrane Proteases, Volume 584

• Preface
• 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
• Author Index
• Subject Index

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.

• 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