Pore-Forming Toxins

1st Edition, Volume 649 - March 10, 2021
Editor: Alejandro Heuck
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 2 3 8 5 8 - 5
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 2 3 8 5 9 - 2

Pore Forming Toxins, Volume 649 in the Methods in Enzymology series continues the legacy of this premier serial with quality chapters authored by field leaders. Chapters in this n… Read more

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Pore Forming Toxins, Volume 649 in the Methods in Enzymology series continues the legacy of this premier serial with quality chapters authored by field leaders. Chapters in this new release include X-ray crystallography shines a light on pore-forming toxins, Giant unilamellar vesicles for studying activity of pore forming proteins, Combined applications of fluorescence spectroscopy and molecular dynamics simulations in studies of Diphtheria Toxin translocation domain, Biophysical approaches to study actinoporin-lipid interactions, Molecular basis for activation of Actinoporins by lipids, Engineered ClyA for detection of biological molecules, Pore-forming Toxins for the Size-Discrimination of Polymers and Biopolymers: Towards Biomolecules Sequencing, and much more.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Chapter One: X-ray crystallography shines a light on pore-forming toxins
Abstract
1: Introduction
2: α-PFTs
3: β-PFTs
4: Conclusion
Acknowledgments
Chapter Two: How protein engineering has revealed the molecular mechanisms of pore-forming toxins
Abstract
1: Introduction
2: Alanine or cysteine scanning of bacterial PFTs
3: The introduction of probes using the cysteine sulfhydryl and their uses
4: Engineering disulfide bridges into PFTs
5: Insights into the pore formatting mechanism by altering the amino acid side-chain properties
6: Concluding remarks
Acknowledgments
Chapter Three: Electron microscopy as a critical tool in the determination of pore forming mechanisms in proteins
Abstract
1: Introduction
2: Materials and equipment
3: Strategies for the application of cryo-EM to pore-forming proteins
4: Quantification and statistical analysis
5: Optimization and troubleshooting
6: Conclusion
Chapter Four: Going full circle: Determining the structures of complement component 9
Abstract
1: Introduction
2: Sources of C9
3: Structural applications of purified C9 protein
4: Summary
Chapter Five: Preparation of Clostridium perfringens binary iota-toxin pore complex for structural analysis using cryo-EM
Abstract
1: Introduction
2: Sample preparation for single-particle analysis
3: Character of membrane-spanning B-component pore
4: Formation of Ia-bound Ib-pore
5: Preparation of iota-toxin pore complex
6: Protocol
7: Summary
Acknowledgments
Chapter Six: AFM imaging of pore forming proteins
Abstract
1: Introduction
2: Protein requirements
3: Supported lipid bilayer preparation
4: The atomic force microscope
5: Imaging of pore forming proteins
6: Data analysis
7: Imaging artefacts and sample defects
8: Complementarity to other methods
Acknowledgments
Chapter Seven: High-speed atomic force microscopy to study pore-forming proteins
Abstract
1: Introduction
2: Materials and equipment
3: Methods
4: Summary
5: Notes
Acknowledgments
Chapter Eight: The use of giant unilamellar vesicles to study functional properties of pore-forming toxins
Abstract
1: Giant unilamellar vesicles (GUVs) as a membrane model system
2: Methods for GUVs formation
3: Electroformation of GUVs
4: Droplet transfer method
5: Monitoring binding of PFTs to GUVs
6: Analyzing permeabilization activity of PFTs by using GUvs
7: Summary and conclusions
Acknowledgment
Chapter Nine: Preparation and utility of asymmetric lipid vesicles for studies of perfringolysin O-lipid interactions
Abstract
1: Introduction
2: Studies of the effect of lipid composition upon PFO binding and PFO domain localization using symmetric vesicles
3: Methods for preparing asymmetric vesicles using cyclodextrins
4: Differences in lipid binding and conformation of PFO in symmetric and asymmetric vesicles
5: Remaining questions about the behavior of PFO in membranes
6: Asymmetric large unilamellar vesicle (aLUVs) preparation procedure using HPαCD
7: aLUVs preparation procedure using MαCD
8: Notes
Acknowledgments
Chapter Ten: Molecular basis for the activation of actinoporins by lipids
Abstract
1: Introduction
2: Purification and hemolysis
3: Interaction of actinoporins with liposomes
4: Crystallization of the pore of FraC
5: Regeneration of monomeric water-soluble protein from transmembrane pores
6: Summary
Chapter Eleven: Biophysical approaches to study actinoporin-lipid interactions
Abstract
1: Introduction
2: Hemolysis
3: Preparation of vesicles
4: Release of aqueous contents from model lipid vesicles
5: Surface plasmon resonance (SPR)
6: Isothermal titration calorimetry (ITC)
7: Evaluating the effect of actinoporin binding on SM-clustering
8: Measuring the effect of actinoporin binding on lipid distribution
9: Membrane binding of proteins based on Trp emission
Chapter Twelve: Conformational switching, refolding and membrane insertion of the diphtheria toxin translocation domain
Abstract
1: Introduction
2: Methods for studies of kinetics and thermodynamics of protein-membrane interactions
3: Methods for studies of structure and topology of membrane-inserted proteins
4: Perspectives
Acknowledgments
Chapter Thirteen: Cysteine-based crosslinking approach for characterization of oligomeric pore-forming proteins in the mitochondrial membranes
Abstract
1: Introduction
2: Materials and equipment
3: Protocol
4: Results and discussion
5: Advantages
6: Limitations and alternatives
Acknowledgments
Chapter Fourteen: Topological analysis of type 3 secretion translocons in native membranes
Abstract
1: Introduction
2: Topology analysis of the translocator PopD in cellular membranes: Experimental rationale
3: Analysis of protein segments exposed into the cytosol of HeLa cells for membrane-inserted translocators
4: Analysis of protein segments exposed to the outside of the HeLa cell plasma membrane
5: Summary
Acknowledgment
Chapter Fifteen: Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers
Abstract
1: Introduction
2: Context: Choosing the right technique
3: Single-molecule methods
4: Imaging modalities
5: Single-molecule imaging of pore-forming toxins in droplet interface bilayers
6: Potential limitations and problems
7: Conclusion
Chapter Sixteen: Using multiscale molecular dynamics simulations to obtain insights into pore forming toxin mechanisms
Abstract
1: Introduction
2: All-atom MD simulations of PFTs
3: MARTINI coarse-grained MD simulations of PFTs
4: Structure-based models for capturing the membrane-triggered conformational transition of an α-PFT monomer
5: Summary
Chapter Seventeen: The use of pore-forming toxins to image lipids and lipid domains
Abstract
1: Introduction
2: Key resources table
3: Preparation of NT-Lys
4: Labeling control and bacterial sphingomyelinase (SMase)-treated HeLa cells with Alexa 647-NT-Lys
5: Intracellular labeling with fluorescent-NT-Lys
6: Alexa647-NT-Lys labeling of HeLa cells for PALM/dSTORM
7: Labeling ultracryo sections with nakanori by immunoelectron microscopy
8: Sodium dodecyl sulfate-digested freeze fracture replica labeling (SDS-FRL) with MBP-lysenin
9: Summary
Acknowledgments
Chapter Eighteen: The use of anthrolysin O and ostreolysin A to study cholesterol in cell membranes
Abstract
1: Introduction
2: Purification of recombinant cholesterol-binding toxin proteins
3: Using toxins as cholesterol sensors on live cells
4: Using toxins to visualize cholesterol accessibility on PM
5: Using toxins to study cholesterol sensing and transport
6: Summary
Acknowledgments
Chapter Nineteen: Strategies for enzymological studies and measurements of biological molecules with the cytolysin A nanopore
Abstract
1: Introduction: The ClyA nanopore
2: The experimental setup
3: How to obtain a single channel
4: Enzymological and protein-ligand binding studies and their signals
5: The effect of electrophoretic forces and nanopore confinement
6: Employing proteins for quantitative measurements of small molecules in complex samples
Chapter Twenty: Pore-forming toxins as tools for polymer analytics: From sizing to sequencing
Abstract
1: Introduction
2: Method for nanopore measurements
3: Data analysis of nanopore recordings
4: Selected examples of Np-SMSD
5: Conclusion and perspectives
Chapter Twenty-One: Use of pore-forming toxins to study co-translocational protein folding
Abstract
1: Introduction
2: The translocating protein substrate
3: The oligonucleotide tagging molecules
4: Protein substrate
5: α-HL nanopore preparation
6: Rotaxane formation
7: Summary
Acknowledgments

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Alejandro Heuck