This detailed volume explores experimental strategies and protocols for the expression, assembly, characterization, and exploitation of transmembrane β-barrel proteins. Beginning with methodologies to study their assembly, the book continues with protocols for characterizing the landscape of transmembrane β-barrel protein interactions with other cellular factors, dissecting processes of protein transport in bacteria and mitochondria, examining structural characterization, determination, and prediction, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Transmembrane β-Barrel Proteins: Methods and Protocols serves as an ideal guide for researchers seeking to expand our knowledge of these vital membrane-spanning proteins.

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Transmembrane -Barrel Proteins: Methods and Protocols (Methods in Molecular Biology Book 2778)

Transmembrane β-Barrel Proteins: Methods and Protocols

Raffaele Ieva

Humana

United States, United States of America

1st ed. 2024, PT, 2024

PS, 2024

Preface
Contents
Contributors
Chapter 1: The Name Is Barrel, β-Barrel
Abbreviations
1 Introduction to β-Barrel Outer Membrane Proteins
2 β-Barrel Architecture
3 Bacterial β-Barrel Functional Groups
3.1 Nonspecific Diffusion Channels
3.2 Specific Diffusion Channels
3.3 Transporters, Autotransporters, and Efflux Pumps
3.4 Adhesins/Virulence Proteins
3.5 Assembly β-Barrel OMPs
3.6 Enzymes
4 Mitochondrial Outer Membrane β-Barrels
5 Chloroplast Outer Membrane β-Barrels
6 β-Barrel Biogenesis
6.1 Bacterial Outer Membrane β-Barrel Biogenesis
6.2 Bacterial Assembly Models
6.2.1 BamA-Passive Models
6.2.2 BamA-Active Models
6.2.3 Chaperone-Assisted Model
6.3 Mitochondrial Outer Membrane β-Barrel Biogenesis
7 β-Barrel Protein Evolution
8 Methodology for Study of β-Barrels
8.1 X-Ray Crystallography and NMR
8.2 Cryo-EM
8.3 Machine Learning Algorithms
9 β-Barrels in Medicine
10 Building Novel Barrels
11 Conclusions and Future Perspectives
References
Chapter 2: Recombinant Expression and Overproduction of Transmembrane β-Barrel Proteins
1 Introduction
2 Materials
2.1 Equipment
2.2 Expression in the Native Membrane
2.3 Expression in Inclusion Bodies
3 Methods
3.1 Expression in the Native Membrane
3.2 Expression in Inclusion Bodies
4 Notes
References
Chapter 3: Bacterial Envelope Fractionation
1 Introduction
2 Materials
2.1 Bacterial Storage, Growth, and Cultivation
2.2 Cell Lysis
2.3 Total Membrane Harvesting, Solubilization, and Sucrose-Based Enrichment
3 Methods
3.1 Bacterial Growth and Cultivation
3.2 Cell Lysis
3.3 Total Membrane Harvest
3.4 Selective Detergent Solubilization
3.5 Sucrose-Gradient-Based Fractionation
3.6 Analysis and Characterization of Membrane Fractions
4 Notes
References
Chapter 4: Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System
1 Introduction
2 Materials
2.1 SpyCatcher-sfGFP Production and Purification
2.2 Fluorescence Measurements
2.3 Fluorescence Microscopy
3 Methods
3.1 Production and Purification of SpyCatcher-sfGFP
3.2 Quantifying Surface Exposure of Intimin by Fluorescence Using SpyCatcher-sfGFP
3.3 Observing Surface Exposure of Intimin by Fluorescence Microscopy Using SpyCatcher-sfGFP
4 Notes
References
Chapter 5: In Vitro Reconstruction of Bacterial β-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Memb...
1 Introduction
2 Materials
2.1 EMM Preparation
2.2 Synthesis of Substrate OMPs
2.2.1 In Vitro Transcription
2.2.2 In Vitro Translation
2.3 EMM Assembly Assay
2.4 Analysis of Assembled OMPs
2.4.1 Analysis of OmpA Assembly
2.4.2 Analysis of EspP Assembly
2.4.3 Analysis of Hia Assembly
2.4.4 Analysis of Hia Assembly by Carbonate Extraction
2.4.5 Analysis of OmpF Assembly
3 Method
3.1 EMM Preparation
3.2 Synthesis of Substrate OMPs
3.2.1 In Vitro Transcription
3.2.2 In Vitro Translation
3.3 EMM Assembly Assay
3.4 Analysis of Assembled OMPs
3.4.1 Analysis of OmpA Assembly Reactions
3.4.2 Analysis of EspP Assembly Reactions
3.4.3 Analysis of Assembled Hia Trimers
3.4.4 Analysis of Hia Assembly by Carbonate Extraction
3.4.5 Analysis of OmpF Assembly
4 Notes
References
Chapter 6: Examining Protein Translocation by β-Barrel Membrane Proteins Using Reconstituted Proteoliposomes
1 Introduction
2 Materials
2.1 Liposome Generation
2.2 Membrane Protein Insertion
2.3 Sucrose Flotation Assay
2.4 Spheroplast Release Assay
2.5 Proteinase K Digestion Assay
3 Methods
3.1 Liposome Preparation
3.2 Membrane Protein Insertion
3.3 Sucrose Flotation Assay (Optional)
3.4 (Lipo)protein Translocation by Reconstituted Proteoliposomes
3.4.1 Substrates Released Directly from Spheroplasts (Semi-In Vitro Translocation Assay, Fig. 4)
3.4.2 Purified Substrates (Full In Vitro Translocation Assay, Fig. 5)
3.5 Assessing (Lipo)protein Translocation by Proteinase K Digestion Assay (Fig. 6)
4 Notes
References
Chapter 7: In Vivo Disulfide-Bond Crosslinking to Study β-Barrel Membrane Protein Interactions, Dynamicity, and Folding Interm...
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Bacterial Disulfide Crosslinking
3.1.1 Preparation of Test Bacteria
3.1.2 Oxidation Assay
3.1.3 SDS-PAGE
3.1.4 Western Immunoblotting
3.1.5 Imaging, Quantitation, and Calculation of Crosslinking Levels
3.2 Kinetics of Disulfide-Bond Formation
3.2.1 Time-Resolved Oxidation Assay
3.3 Disulfide-Bond Assay Coupled with Protein Cell-Surface Accessibility to Protease Treatment
3.3.1 Digestion-Oxidation Assays
3.3.2 Oxidation-Digestion Assays
3.4 Disulfide-Bond Assay Coupled with Heat-Induced Mobility Shift
3.4.1 Oxidation Assay with Modified Lysis
3.4.2 Cold-SDS-PAGE
4 Notes
References
Chapter 8: Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial β-Barrel Assembly Machine
1 Introduction
2 Materials
2.1 Protein Expression Strain and Plasmids
2.2 Bacterial Growth, Protein Expression, and Cell Lysis
2.3 Buffers
2.4 Protein Purification and Quantification
2.5 Photocrosslinking
2.6 SDS-PAGE and Immunoblotting
3 Methods
3.1 Expression and Purification of His-CdiA Receptor-Binding Domain (CdiA RBD)
3.2 Expression and Purification of BamA with pBPA Substitutions
3.3 Purification of BamA-CdiA RBD Complexes
3.4 Photocrosslinking of BamA-CdiA RBD Complexes
3.5 SDS-PAGE and Immunoblotting
4 Notes
References
Chapter 9: Analysis of Transmembrane β-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis
1 Introduction
2 Materials
2.1 BN-PAGE Analysis of Transmembrane β-Barrel Protein Complexes
2.1.1 BN Polyacrylamide Gradient Gel Preparation
2.1.2 Preparation of BN-PAGE Samples
2.1.3 BN Gel Staining
2.2 Heat Modifiability Assay
2.2.1 Bacterial Strains and Expression Plasmids
2.2.2 Bacterial Growth and Protein Expression
2.2.3 Semi-native PAGE
2.2.4 Proteins Immuno-Detection
3 Methods
3.1 BN-PAGE Assay
3.1.1 Preparation of BN Polyacrylamide Gradient Gels
3.1.2 Preparation of Protein Sample for Migration on BN-PAGE
3.1.3 Migration of Proteins by BN-PAGE
3.1.4 Staining of BN Gels
3.2 Heat Modifiability Assay
3.2.1 Overexpression of OmpA and BamA either Alone or with DolP and Preparation of Total Protein Extracts
3.2.2 Loading and Migration on Semi-native PAGE
3.2.3 Protein Transfer to a PVDF Membrane
3.2.4 Immunodetection
4 Notes
References
Chapter 10: Affinity Purification of Membrane β-Barrel Proteins via Biotin-Tagged Peptidiscs
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Crude Membrane Preparation
2.3 Membrane Solubilization
2.4 Peptidisc Library Reconstitution and Purification
2.5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.6 Mass Spectrometry Sample Preparation
2.7 Protein Identification
3 Methods
3.1 Cell Growth and Crude Membrane Preparation
3.2 Outer Membrane Solubilization
3.3 Peptidisc Library Reconstitution and Purification
3.4 SDS-PAGE and In-Gel Digestion
3.5 In-Solution Digestion
3.6 Peptides Desalting Using C18 Stage Tips
3.7 Protein Identification (See Note 19)
3.8 Data Analysis
4 Notes
References
Chapter 11: Monitoring the Interaction of the Peptidoglycan with the Bacterial β-Barrel Assembly Machinery
1 Introduction
2 Materials
2.1 Protein Purification
2.2 Ni-NTA Affinity Chromatography
2.3 Size-Exclusion Chromatography
2.4 Preparation of PG Sacculi and Soluble PG Fragments
2.5 Generation of Tetran Chains
2.6 PG Pull-Down Assay
2.7 Microscale Thermophoresis Experiments
2.8 SDS-Denaturation Test for Microscale Thermophoresis
2.9 In Vivo Cross-linking of Bam Proteins to PG
3 Methods
3.1 Protein Purification
3.1.1 Purification of Bam Proteins
3.1.2 Purification of BamABCDE
3.1.3 Purification of MepM DD-endopeptidase to Generate Soluble PG Fragments for Bam-PG Interaction Studies
3.2 Preparation of PG Sacculi and Soluble PG Fragments for Protein-PG Interaction Assays
3.2.1 PG Isolation from E. coli
3.2.2 Hayashi ́s Test for the Detection of SDS During PG Isolation
3.2.3 Generation of Tetran Chains as Ligands for the Bam Proteins in Microscale Thermophoresis Experiments
3.3 BAM-PG Interaction Assays
3.3.1 PG Pull-Down Assay
3.3.2 Microscale Thermophoresis Experiments to Detect the Interaction Between Purified Bam Proteins and Tetran PG Chains
3.3.3 SDS-Denaturation Test to Validate Ligand-Dependent Changes in Initial Fluorescence During Microscale Thermophoresis Expe...
3.3.4 In Vivo Cross-linking of Bam Proteins to PG
4 Notes
References
Chapter 12: MitoLuc: A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria
1 Introduction
2 Materials
2.1 Producing Yeast That Constitutively Express Mitochondrial Matrix-Targeted 11S
2.2 Isolation of Mitochondria from Yeast Cells with Matrix-Localized 11S
2.3 Expression and Purification of GST-Dark, GST-rPFO (Recombinant Perfringolysin O), and pep86-Precursor Fusion Proteins
2.4 NanoLuc-Based Import Assay with Isolated Yeast Mitochondria
2.5 NanoLuc-Based Import Assay in Mammalian Cells (MitoLuc Import Assay)
3 Methods
3.1 Generating a Yeast Cell Line That Constitutively Expresses Mitochondrial Matrix-Targeted 11S
3.2 Isolation of Mitochondria from YPH499-mt-11S Yeast Cells
3.3 Expression and Purification of GST-Dark, GST-rPFO (Recombinant Perfringolysin O), and pep86-Precursor Fusion Proteins
3.4 NanoLuc Assay to Measure Protein Import into Isolated Yeast Mitochondria
3.5 NanoLuc Assay to Measure Protein Import in Permeabilized Mammalian Cells (MitoLuc Assay)
4 Notes
References
Chapter 13: Modular Assembly of Mitochondrial β-Barrel Proteins
1 Introduction
2 Materials
2.1 In Vitro Transcription
2.2 Cell-Free Protein Synthesis
2.3 Isolation of Mitochondria
2.4 Protein Import Assay into Isolated Mitochondria
2.5 Blue Native Electrophoresis
2.6 Proteinase K Treatment
2.7 Carbonate Extraction
2.8 Import of Chemical Amounts of β-Barrel Proteins
3 Methods
3.1 In Vitro Transcription
3.2 Cell-Free Protein Synthesis
3.2.1 In Vitro Synthesis of Radiolabelled Proteins with the Coupled Transcription/Translation Reaction
3.2.2 In Vitro Synthesis of Radiolabelled Proteins with the Translation Reaction Kit
3.3 Isolation of Mitochondria
3.4 Protein Import Assay into Isolated Mitochondria
3.5 Blue Native Electrophoresis
3.6 Proteinase K Treatment
3.7 Carbonate Extraction
3.8 Import of Chemical Amounts of β-Barrel Proteins
4 Notes
References
Chapter 14: Tracking the Activity and Position of Mitochondrial β-Barrel Proteins
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Fabrication of Agarose-Supported Droplet-Interface Bilayers
3.1.1 Lipid Preparation
3.1.2 Formation of Lipid Monolayers at the Oil-Water Interface Around Aqueous Droplets
3.1.3 PMMA Chamber Assembly and Formation of a Lipid Monolayer at the Oil-Hydrogel Interface
3.1.4 Formation of DIB Membranes
3.2 Recording DIB Images
3.3 Data Analysis
3.3.1 Background Correction
3.3.2 Tracking Individual Channels
4 Notes
References
Chapter 15: Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulse...
1 Introduction
2 Materials
2.1 Site-Directed Mutagenesis and Transformation
2.2 Protein Expression
2.3 Isolation of Outer Membrane
2.4 Spin Labeling in Outer Membrane and E. coli
2.5 Equipment
2.6 Software Packages
3 Methods
3.1 Plasmid Construction and Site-Directed Mutagenesis
3.2 Transformation
3.3 Protein Expression
3.3.1 Inducible Expression of BamABCDE
3.3.2 Constitutive Expression of BtuB
3.4 Isolation of Outer Membrane
3.5 Spin Labeling in Outer Membrane
3.6 Spin Labeling in E. coli
3.7 CW ESR
3.8 PELDOR
3.9 PELDOR Data Analysis and Interpretation
4 Notes
References
Chapter 16: Characterization of β-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinki...
1 Introduction
2 Materials
2.1 Protein Samples
2.2 Crosslinking
2.3 In-Gel Digestion
2.4 Liquid Chromatography-Mass Spectrometry
2.5 Data Analysis and Visualization
3 Methods
3.1 Crosslinking
3.2 In-Gel Digestion
3.3 Liquid Chromatography-Mass Spectrometry
3.4 Data Analysis and Visualization
4 Notes
References
Chapter 17: Dissecting the Organization of a β-Barrel Assembly Machinery (BAM) Complex by Neutron Reflectometry
1 Introduction
2 Materials
3 Methods
3.1 Nickel-Nitrilotriacetic Acid (Ni-NTA) Modification
3.2 NR Cell Cleaning and Assembling
3.3 BamA Assembly on Ni-NTA Surface
3.4 Removal of DDM
3.5 Introduction of Supported Lipid Bilayer
3.6 Formation of BamAB Architecture
3.7 Formation of BamABDE Architecture
3.8 Data Analysis
4 Notes
References
Chapter 18: Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel
1 Introduction
2 Materials
2.1 Expression of His-Tagged pIV Protein
2.2 Small-Scale Comparison of Detergents for Optimal Solubilization of the pIV Secretin
2.3 Large-Scale Purification of pIV Protein Using CHAPS Detergent
2.4 Electron Microscopy
3 Methods
3.1 Expression of His-Tagged pIV Protein (See Note 9)
3.2 Small-Scale Comparison of Detergents for Optimal Solubilization of the pIV Secretin
3.2.1 TCA Precipitation for SDS-PAGE and Western Blotting
3.2.2 Preparation of ``Cold Competitor Lysate ́ ́ for Western Blotting
3.2.3 Western Blotting
3.3 Large-Scale Purification of pIV Protein Using CHAPS Detergent
3.4 Electron Microscopy
3.4.1 Negative Stain - Grid Preparation and Observation
3.4.2 Cryo-EM Grid Preparation
3.4.3 Cryo-EM Data Collection
3.4.4 Cryo-EM Image Processing
3.4.5 Model Building and Refinement
4 Notes
References
Chapter 19: Recent Advances in Modeling Membrane β-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Envi...
Abbreviations
1 Introduction
2 Methodological Development to Model β-Barrel Proteins
3 Through the Pore: From Channels to Nanopores
4 β-Barrel Proteins in Their Local Environment
5 Biogenesis and Large Assemblies of β-Barrel Proteins
6 Beyond the Membrane Plane
7 Conclusion
8 Notes
References
Chapter 20: Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes
1 Introduction
2 Materials
2.1 Computational
2.1.1 In Silico Modeling
2.1.2 MD Simulations and Analysis
2.2 Bacterial
2.2.1 PorV Expression
2.2.2 PorV:RgpB-CTD Surface Binding Assay
3 Methods
3.1 Computational
3.1.1 In Silico Modeling
3.1.2 MD Simulations
3.1.3 MD Analysis
3.2 Bacterial
3.2.1 PorV Expression
3.2.2 PorV:RgpB-CTD Surface Binding Assay
4 Notes
References
Chapter 21: Rationale in Custom Design of Transmembrane β-Barrel Pores
1 Introduction
2 Materials
3 Methods
3.1 Blueprint Design of the β-Barrel Backbones
3.1.1 Define Backbone Architecture
3.1.2 Transmembrane Span
3.1.3 Blueprint Generation
3.1.4 Sculpting the β-Barrel with Torsional Irregularities
3.1.5 Explicit Assembly of β-Barrel Backbones
3.2 Placing the Mortise-Tenon Folding Motifs
3.3 Design of TMB Amino Acid Sequences
3.4 Final Selection of Designs for Experimental Characterization
4 Notes
References
Chapter 22: Stress-Based Screening for Compounds That Inhibit β-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacter...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Bacterial Strains
2.3 M9 Medium
2.4 Compound Stocks
2.5 Equipment
3 Methods
3.1 Screening a Compound Library
3.2 Dose-Response Kinetics and Orthogonal Stress Responses
4 Notes
References
Index

2024-03-23