Transmembrane ß-Barrel Proteins Methods and Protocols

This detailed volume explores experimental strategies and protocols for the expression, assembly, characterization, and exploitation of transmembrane ß-barrel proteins. Beginning with methodologies to study their assembly, the book continues with protocols for characterizing the landscape of transmembrane ß-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. 

The Name is Barrel, ß-Barrel.- Recombinant Expression and Overproduction of Transmembrane b-Barrel Proteins.- Bacterial Envelope Fractionation.- Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System.- In Vitro Reconstruction of Bacterial ß-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.- Examining Protein Translocation by ß-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.- In Vivo Disulfide-Bond Crosslinking to Study ß-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.- Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial ß-Barrel Assembly Machine.- Analysis of Transmembrane ß-Barrel Proteins by Native and Semi-Native Polyacrylamide Gel Electrophoresis.- Affinity Purification of Membrane ß-Barrel Proteins via Biotin-Tagged Peptidiscs.- Monitoring the Interaction of the Peptidoglycan with the Bacterial ß-Barrel Assembly Machinery.- MitoLuc:A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria.- Modular Assembly of Mitochondrial ß-Barrel Proteins.- Tracking the Activity and Position of Mitochondrial ß-Barrel Proteins.- Conformational Heterogeneity of ß-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.- Characterization of ß-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinking Mass Spectrometry.- Dissecting the Organization of a ß-Barrel Assembly Machinery (BAM) Complex by Neutron Reflectometry.- Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel.- Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.- Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.- Rationale in Custom Design of Transmembrane ß-Barrel Pores.- Stress-Based Screening for Compounds that Inhibit ß-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacteria.