As the mysteries stored in our DNA have been more completely revealed, scientists have begun to face the extraordinary challenge of unraveling the int- cate network of proteinprotein interactions established by that DNA fra- work. It is increasingly clear that proteins continuously interact with one another in a highly regulated fashion to determine cell fate, such as proliferation, diff- entiation, or death. These proteinprotein interactions enable and exert str- gent control over DNA replication, RNA transcription, protein translation, macromolecular assembly and degradation, and signal transduction; essentially all cellular functions involve proteinprotein interactions. Thus, proteinp- tein interactions are fundamental for normal physiology in all organisms. Alt- ation of critical proteinprotein interactions is thought to be involved in the development of many diseases, such as neurodegenerative disorders, cancers, and infectious diseases. Therefore, examination of when and how proteinp- tein interactions occur and how they are controlled is essential for understa- ing diverse biological processes as well as for elucidating the molecular basis of diseases and identifying potential targets for therapeutic interventions. Over the years, many innovative biochemical, biophysical, genetic, and computational approaches have been developed to detect and analyze p- teinprotein interactions. This multitude of techniques is mandated by the diversity of physical and chemical properties of proteins and the sensitivity of proteinprotein interactions to cellular conditions.

Includes supplementary material: sn.pub/extras

Klappentext
Proteins continuously interact with each other to determine cell fate. Consequently, an examination of just when such protein-protein interactions occur and how they are controlled is essential for understanding the molecular mechanism of biological processes, elucidating the molecular basis of diseases, and identifying potential targets for therapeutic interventions. In Protein-Protein Interactions: Methods and Applications, leading experts describe in detail their highly successful biochemical, biophysical, genetic, and computational techniques for studying these interactions. Their readily reproducible methods demonstrate how to identify protein interaction partners, qualitatively or quantitatively measure protein-protein interactions, monitor protein-protein interactions as they occur in living cells, and determine interaction interfaces. The techniques described utilize a variety of cutting-edge technologies, including surface plasmon resonance (SRP), fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), isothermal titration calorimetry (ITC), circular dichroism (CD), protein fragment complementation assays (PCA), various two-hybrid systems, and proteomics- and bioinformatics-based approaches, such as the Scansite program for computational analysis. Each time-tested protocol includes a background introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and highly practical, Protein-Protein Interactions: Methods and Applications offers both beginning and experienced investigators a full range of the powerful tools needed for deciphering how proteins interact to form biological networks, as well as for unraveling protein-protein interactions in disease in the search for novel therapeutic targets.

Inhalt
Overview.- Structural Basis of Protein-Protein Interactions.- Quantitative Analysis of Protein-Protein Interactions.- In Vitro Techniques.- Characterization of Protein-Protein Interactions by Isothermal Titration Calorimetry.- Circular Dichroism Analysis for Protein-Protein Interactions.- Protein-Protein Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy.- Measuring Rhodopsin-G-Protein Interactions by Surface Plasmon Resonance.- Using Light Scattering to Determine the Stoichiometry of Protein Complexes.- Sedimentation Equilibrium Studies.- Analysis of Protein-Protein Interactions by Simulation of Small-Zone Gel Filtration Chromatography.- Fluorescence Gel Retardation Assay to Detect Protein-Protein Interactions.- Fluorescence Polarization Assay to Quantify Protein-Protein Interactions.- Studying Protein-Protein Interactions via Blot Overlay or Far Western Blot.- Glutathione-S-Transferase-Fusion Based Assays for Studying Protein-Protein Interactions.- Affinity Capillary Electrophoresis Analyses of ProteinProtein Interactions in Target-Directed Drug Discovery.- Mapping Protein-Ligand Interactions by Hydroxyl-Radical Protein Footprinting.- Use of Phage Display and Polyvalency to Design Inhibitors of Protein-Protein Interactions.- Detecting Protein-Protein Interactions in Heterologous Systems.- A Bacterial Two-Hybrid System Based on Transcription Activation.- Using the Yeast Two-Hybrid System to Identify Interacting Proteins.- Analysis of Protein-Protein Interactions Utilizing Dual Bait Yeast Two-Hybrid System.- The Split-Ubiquitin Membrane-Based Yeast Two-Hybrid System.- Reverse Two-Hybrid Techniques in the Yeast Saccharomyces cerevisiae.- Mammalian Two-Hybrid Assay for Detecting Protein-Protein Interactions In Vivo.- Co-Immunoprecipitation from TransfectedCells.- Probing Protein-Protein Interactions in Living Cells.- Microscopic Analysis of Fluorescence Resonance Energy Transfer (FRET).- Monitoring Molecular Interactions in Living Cells Using Flow Cytometric Analysis of Fluorescence Resonance Energy Transfer.- Fluorescence Correlation Spectroscopy.- Confocal Microscopy for Intracellular Co-Localization of Proteins.- Mapping Biochemical Networks With Protein-Fragment Complementation Assays.- In Vivo Protein Cross-Linking.- Proteomics-Based Approaches.- Computational Prediction of Protein-Protein Interactions.- Affinity Methods for Phosphorylation-Dependent Interactions.- Two-Dimensional Gel Electrophoresis for Analysis of Protein Complexes.- Sample Preparation of Gel Electrophoretically Separated Protein Binding Partners for Analysis by Mass Spectrometry.- Quantitative Protein Analysis by Solid Phase Isotope Tagging and Mass Spectrometry.- Internet Resources for Studying Protein-Protein Interactions.