Multiprotein Complexes Methods and Protocols

This volume explores strategies and detailed protocols for the preparation of macromolecular complexes and their characterization in view of structural analysis. The chapters in this book are separated into three parts: Part One focuses on sample preparation, and covers strategies for recombinant expression of multiprotein complexes in prokaryotic and eukaryotic hosts, for genome engineering using the CRISPR/Cas9 system and for production of specific binders such as reformatted antibodies and artificial binding proteins. Part Two looks at the biophysical methods that can provide useful indicators for sample optimization, and often complement structural information obtained with core technologies for structure determination—x-ray crystallography and cryo-electron microscopy—by quantitative solution data. Part Three discusses the characterization of multiprotein complexes in a cellular environment using the latest technologies and in vivo approaches. Written in the highly successfulMethods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and authoritative, Multiprotein Complexes: Methods and Protocols is a valuable resource for structural and molecular biologists who need to prepare multi-components for their applications, and for other scientists working on macromolecular assemblies from other angles that need to know the latest approaches that the field has to offer.

Production of Multi-Subunit Membrane Protein Complexes.- Production of Multiprotein Complexes using the Baculovirus Expression System: Homology Based and Restriction Free Cloning Strategies for Construct Design.- Tagging Proteins with Fluorescent Reporters using the CRISPR/Cas9 System and Double Stranded DNA Donors.- Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System.- The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments.- Isolation of Artificial Binding Proteins (Affimer Reagents) for Use in Molecular and Cellular Biology.- Interactions Measurements of Protein-DNA Complexes by Isothermal Titration Calorimetry (ITC) and Microscale Thermophoresis (MST).- switchSENSE Technology for Analysis of DNA Polymerase Kinetics.- Sedimentation Velocity Methods for Characterization of Protein Heterogeneity and Protein Affinity Interactions.- Hands on Native Mass Spectrometry Analysis of Multi-Protein Complexes.- Studying Protein-DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry.- Integrative Mass Spectrometry-Based Approaches for Modeling Macromolecular Assemblies.- Optimization of Sample Preparation for the Observation of Macromolecular Complexes by Electron (cryo-)Microscopy.- Solubilization and Stabilization of Native Membrane Proteins for Drug Discovery.- Practical Aspects of Super-Resolution Imaging and Segmentation of Macromolecular Complexes by dSTORM.- Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in Live Cells.- Context-Specific and Proximity-Dependent Labeling for the Proteomic Analysis of Spatiotemporally Defined Protein Complexes with Split-BioID.- A Directed Evolution System for Lysine Deacetylases.