As more genetic and biochemical information about the protein components of cells accumulate, the analysis of protein-protein interactions is becoming increasingly important. This manual presents a wide range of techniques for identifying and analyzing these interactions, starting with standard molecular and biochemical techniques, and progressing to biophysical and computational approaches and therapeutic and other post-genomic applications. This manual is designed to complement the imformation in the Molecular Cloning manual, and is presented in the same clear, user-friendly format. It should be a useful resource for investigators studying interacting sets of proteins and their physiologic significance in a wide range of experimental systems.

Section 1 Protein-Protein Interactions in Biological Context

1.Protein-Protein Interactions: A Common Theme in CeU Biology, 1

2.Building a Better Web: Progress in the Concept and Methodology of Protein Interaction Studies, 13

Section 2 Standard Technologies to Probe Protein Interactions

3.Identification of Protein-Protein Interactions by Conventional Column Chromatography, 37

4.Identification of Associated Proteins by Coimmunoprecipitation, 55

5.Chromatin Immunoprecipitation of Protein Complexes, 67

6.Identification of Protein-Protein Interactions with Glutathione-S-Transferase Fusion Proteins, 81

7.Protein Interactions Captured by Chemical Cross-linking, 105

8. Identification of Protein-Protein Interactions by Overlay Screening of Phage cDNA EXpression Libraries, 129

9.Phage-display Methodology for the Study of Protein-Protein Interaction,14l

10.Yeast Two-Hybrid System for Studying Protein-Protein Interactions, 167

11. Bacterial Two-Hybrid System for Studying and Modifying Protein-Protein Interactions, 195

12. Genetic Approaches to Identify Protein-Protein Interactions in Budding Yeast, 217

Section 3 Biophysical Approaches to Probe Protein Interactions

13.Calorimetry-based Approaches in Analysis of Protein-Protein Interactions, 229

14. Analytical Ultracentrifugation in the Study of Protein Self-association and Heterogeneous Protein-Protein Interactions, 253

15. Monitoring Protein-Protein Interactions by Time-resolved FTIR Difference Spectroscopy,279

16. Protein Co-crystallization for X-ray Structure Determination, 301

17. Characterization ofMultiprotein Complexes by Mass Spectrometry, 317

18. Identification of Novel Protein Complexes and Protein-Protein Interactions by Mass Spectrometry, 329

19. Measurement of Protein-Protein Interactions Using Surface Plasmon Resonance Spectroscopy, 355

20. Advancement and Utilization of Biosensors for Protein Interaction Analysis and Proteomics, 377

21. Probing Ligand-Receptor Interactions with Atomic Force Microscopy, 399

22. Analysis of Protein-DNA Interactions by Optical Tweezers: Application to Chromatin Fibers, 415

23. Evanescent Field Fluorescence Microscopy for Analysis of Protein- DNA Interactions at the Single-Molecule Level, 429

Section 4 Novel High-Throught Approaches to Probe Probtein Interactions

24. Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates, 445

25. The Split-Ubiquitin Membrane-based Yeast Two-Hybrid System, 469

26. A Bacterial Two-Hybrid System Based on a cAMP Signaling Cascade, 499

27. In Vitro Selection and Evolution of Protein-Ligand Interactions by Ribosome Display, 517

28. Protein-dependent Ribozymes as Reporters of Protein-Protein Interactions, 549

Section 5 Interactions of Proteins and Peptides

29. Screening Kinase Phosphorylation Motifs Using Peptide Libraries, 561

30. Using Peptide Aptamers to Study Proteins and Protein Networks, 575

31. Analysis of Protein Interactions with Immobilized Peptide Arrays Synthesized on Membrane Supports, 591

Section 6 In Vivo Imaging of Protein Interactions

32. Imaging Protein Interactions by FRET Microscopy, 609

33. Detection of Protein Interactions and Library Screening with Protein-fragment Complementation Assays, 637

34. Direct Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence Complementation Analysis, 673

35. Imaging Protein- Protein Interactions in Living Animals, 695

36. Electron Microscopy Tomography and Localization of Proteins and Macromolecular

Section 7 Genome-wide and Computer-based Analysis of Protein Interactions

37. Genome-wide Analysis of Protein- Protein interactions Using a Two-Hybrid Array,741

38. Tandem Affinity Purification Strategy for Protein Complex Characterization, 761

39. Combining Phage Display and Unrelated Orthogonal Approaches to Determine Interaction Networks Mediated by Protein Recognition Modules, 775

40. Exploiting Synthetic Genetic Interactions to Predict Pathways and Complexes, 797

41. Analysis of Genome-wide Protein Interactions Using Computational Approaches, 813

42. Visualization and Integration of Protein Interaction Networks, 839

Section 8 Design of Novel Interactions and Inhibitors in Drug Discovery

43. Engineering Antibodies with Preferred Antigen Specificity, 857

44. Discovery of a Growth Hormone Antagonist Using a Structure- Function Approach, 873

45. Computational Docking of Biomolecular Complexes with AutoDock, 885

46. Exploiting Protein-Protein Interactions to Design an Activator of p53, 893

Appendix: Cautions, 907

Index, 919

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