ENZYMOLOGY AT THE MEMBRANE INTERFACE: INTRAMEMBRANE PROTEASES. METHODS IN ENZYMOLOGY, VOL. 584

• Chapter One: Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease
o Abstract
o 1 Introduction
o 2 The Use of MBP-Tagged Model Substrates for Analysis of Proteolytic Activity of RseP
o 3 In Vitro Analysis of Proteolysis of SPs by RseP
o 4 Structural Analysis of RseP Using Thiol-Specific Modification Reagents
o 5 In Vivo Cross-Linking Analysis to Probe the Interaction of RseP With Its Substrate in the Membrane
o Acknowledgments
• Chapter Two: Signal Peptidase Enzymology and Substrate Specificity Profiling
o Abstract
o 1 Introduction
o 2 Purification of ?2-75 SP1
o 3 Purification of SP1 Substrate pONA
o 4 In Vitro Signal Peptide Cleavage Assay
o 5 In Vivo Assay of Signal Peptidase Activity
o 6 Substrate Specificity Profiling
o 7 Conclusions
o Acknowledgment
• Chapter Three: Screening and Characterization Strategies for Nanobodies Targeting Membrane Proteins
o Abstract
o 1 Introduction
o 2 Phage Display
o 3 Critical Aspects of Biopannings
o 4 Screening Strategies
o 5 Characterization of Nanobodies
o 6 Potential Applications
o Acknowledgments
• Chapter Four: Probing the Activity of Eukaryotic Rhomboid Proteases In Vitro
o Abstract
o 1 Introduction
o 2 Expression and Solubilization of Rhomboid Proteases From Bacteria
o 3 In Vitro Probing of Rhomboid Protease Activity
o Acknowledgments
• Chapter Five: Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases
o Abstract
o 1 Introduction to Intramembrane Proteases
o 2 Expression and Purification of GlpG, S2P, and PSH
o 3 Enzymatic Activity Assays for Intramembrane Proteases
o 4 Expression and Purification of Human ?-Secretase
o 5 Enzymatic Activity Assay for Human ?-Secretase
o 6 Structures of I-CLiPs
o 7 Concluding Remarks
o Acknowledgments
• Chapter Six: Analyzing Amyloid-ß Peptide Modulation Profiles and Binding Sites of ?-Secretase Modulators
o Abstract
o 1 Introduction
o 2 Methods
o Acknowledgments
• Chapter Seven: Probing the Structure and Function Relationships of Presenilin by Substituted-Cysteine Accessibility Method
o Abstract
o 1 Introduction
o 2 Principle of Cysteine-Based Structural Analyses of Presenilin
o 3 Protocol
o 4 Summary and Perspectives
o Acknowledgments
• Chapter Eight: A New Method to Determine the Transmembrane Conformation of Substrates in Intramembrane Proteolysis by Deep-UV Resonance Raman Spectroscopy
o Abstract
o 1 Introduction
o 2 In Vitro Intramembrane Proteolysis Assay
o 3 Intramembrane Proteolysis of the Substrate Monitored by dUVRR Spectroscopy
o 4 Conclusions
o Acknowledgment
• Chapter Nine: An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane
o Abstract
o 1 Introduction
o 2 Enzymatic Considerations for Kinetic Analysis of Proteolysis in Detergent Micelle Systems
o 3 A New Strategy: Quantitative Analysis in Proteoliposomes
o 4 Preparation of Liposomes
o 5 Preparation of Fluorophore-Labeled Substrate
o 6 Establishing an Inducible and Fluorogenic Intramembrane Protease Assay
o 7 Real-Time Kinetic Analysis of Membrane-Immersed Proteolysis
o 8 Summary
o Acknowledgments
• Chapter Ten: Production of Recombinant Rhomboid Proteases
o Abstract
o 1 Introduction
o 2 Overview
o 3 Example of an Expression and Purification Protocol—The pBAD System
o Acknowledgments
• Chapter Eleven: Mechanism and Inhibition of Rhomboid Proteases
o Abstract
o 1 Introduction
o 2 Rhomboid Protease Mechanism
o 3 Substrate Specificity of Rhomboid Proteases
o 4 Rhomboid Protease Inhibitors
o 5 Concluding Remarks and Perspectives
o Acknowledgments
• Chapter Twelve: Enzymatic Assays for Studying Intramembrane Proteolysis
o Abstract
o 1 Introduction
o 2 Detergent-Solubilized Assay
o 3 Proteoliposome Assay
o 4 Inducible Proteoliposome Assay
o 5 Conclusions
o Acknowledgments
• Chapter Thirteen: Methods for Structural and Functional Analyses of Intramembrane Prenyltransferases in the UbiA Superfamily
o Abstract
o 1 Introduction to the UbiA Superfamily
o 2 Biological Function and Enzymatic Activity of the Subfamilies
o 3 Predicting the Functional Role of Intramembrane Prenyltransferases by Sequence Clustering Analysis
o 4 Analysis of Enzymatic Activity
o 5 Structural Studies of the UbiA Superfamily
o Acknowledgments
• Chapter Fourteen: Functional Study of the Vitamin K Cycle Enzymes in Live Cells
o Abstract
o 1 Introduction
o 2 Cell-Based Assay for Functional Study of Vitamin K Cycle Enzymes
o 3 Functional Study of VKOR Using TALENs-Mediated Gene Knockout Reporter Cells
o 4 Functional Study of GGCX Using CRISPR-Cas9-Mediated Gene Knockout Reporter Cells
o 5 Concluding Remarks
• Chapter Fifteen: Activity Assays for Rhomboid Proteases
o Abstract
o 1 Introduction
o 2 In Vivo Assays
o 3 In Vitro Assays
o Acknowledgments
• Author Index
• Subject Index

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

Author
Michael H Gelb, Dept. of Chemistry, University of Washington, Seattle, USA