About the Book

Dynamic modulation of protein–protein interactions provides the physical basis for many biological signaling networks. Consequently, over the last several decades, much effort has been invested in defining and understanding these interactions. Knowledge of the pattern and regulation of discrete protein–protein interactions, and the larger networks that are built from them, tells us how living organisms function as homeostatic entities in the face of a changing environment and also how they develop and change their phenotypes over time or in response to specific triggers. In addition, characterization of protein–protein interactions informs us of the molecular basis of human disease and provides opportunities to intervene to prevent, detect, and treat disease. The goal of this book is to describe classic and cutting edge techniques to identify, characterize, and modulate simple and complex protein–protein interactions. This manual, which includes discussions and analyses that accompany detailed step-by-step protocols, is presented in eight sections.

Section 1: Protein–Protein Interactions in Biological Context

This section provides two overviews of protein–protein interactions, to set the context for all the technologies discussed in the book. Pawson (1) describes the role of protein–protein interactions in biology and their assembly from modular protein domains. Golemis (2) compares the historical changes in the approaches used to discern a protein's function, through analysis of protein–protein interactions in the pre- and postgenomic eras.

Section 2: Standard Technologies to Probe Protein Interactions

Ten chapters describe varied approaches to identify and analyze protein–protein interactions that have stood the test of time. Sudakin (3) describes isolation of protein complexes by classical column purification techniques. Ohh (4) describes the widely used coimmunoprecipitation technique, with Orlando (5) extending this technique to address chromatin immunoprecipitation. Einarson (6) details common uses of glutathione-S-transferase (GST) technology. Carlson (7) describes the issues associated with chemical cross-linking analysis. Zhao (8) and Silverman (9) present applications of phage-display technology. Serebriiskii (10) and Joung (11) give a detailed account of yeast and bacterial two-hybrid systems, while Strich (12) describes approaches to infer protein interactions from genetic analysis.

Section 3: Biophysical Approaches to Probe Protein Interactions

Eleven chapters detail sophisticated biophysical approaches to probe protein–protein interactions. Some of these methods, such as the mass spectrometry described by Robinson (17) and Yates (18), can be used to identify novel protein–protein interactions. Others, such as the chapters on calorimetry by Herrmann (13) and X-ray crystallography by Marmorstein (16), provide quantitative thermodynamic/energetic or high-resolution structural information on known interactions. In other chapters, Shuck (14) presents analytical ultracentrifugation, Gerwert (15) describes FTIR difference spectroscopy, and Fisher (19) and Chaiken (20) describe the use of surface plasmon resonance alone or in conjunction with other approaches. Moy (21) illustrates atomic force microscopy, Zlatanova (22) details the use of optical tweezers, and Leuba (23) presents evanescent field fluorescence microscopy.

Section 4: Novel High-Throughput Approaches to Probe Protein Interactions

Five chapters describe powerful, but less widely used, approaches to screen for protein–protein interactions. Weber (24) illustrates the recently developed approach to screen for novel protein kinase substrates, using ATP analogs. Plückthun (27) details ribosome display. The latter is one of several chapters in this book that are of note because they facilitate multiple sequential rounds of mutagenesis and selective binding assays to optimize protein function. In other chapters, Stagljar (25) describes a split-ubiquitin membrane-based two-hybrid system, Ladant (26) provides a summary of a bacterial two-hybrid approach based on modulation of cAMP signaling, and Hartig (28) describes protein-directed ribozymes.

Section 5: Interactions of Proteins and Peptides

A frequent goal in analysis of protein–protein interactions is to define the minimum interacting domains of the partners. This section has three chapters covering methods to examine interactions between proteins and peptides, with Yaffe (29) describing the use of peptide libraries to identify kinase phosphorylation motifs, Finley (30) describing the identification and use of peptide aptamers, and Dübel (31) describing probes of immobilized peptide arrays.

Section 6: In Vivo Imaging of Protein Interactions

It is obviously important to understand protein–protein interactions in their physiological environment. These five chapters give accounts of approaches to study protein–protein interactions in an in vivo context. Chapters by Bastiaens (32), Michnick (33), and Kerppola (34) look at approaches to study protein interactions in living cells. The chapter by Gambhir (35) describes methods to examine protein interactions in small living mammals, while Koster (36) describes immunoelectron microscopy approaches to study protein interactions at very high resolution.

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

Six chapters take a contemporary genome- or proteome-wide view of protein–protein interactions. The chapters by Miller (37) and Seraphin (38) take the experimental approach to identify protein–protein interactions throughout the proteome. Cesareni (39) describes the combination of multiple techniques working by distinct approaches to enrich information content of interaction significance. The chapters by Boone (40), Ouzounis (41), and Ideker (42) describe computer-based approaches that either predict novel protein–protein interactions or build interaction networks that chart the complex mesh of dynamic interactions within the cell.

Section 8: Design of Novel Interactions and Inhibitors in Drug Discovery

If the study of protein–protein interactions over the last few decades has a direction or purpose, then the design of therapeutic molecules is clearly a valid endpoint. These four chapters, by Kipryanov (43), Kopchick (44), Goodsell (45), and Vassilev (46), describe how knowledge of protein–protein interactions can be exploited to optimize protein function or rationally design therapeutic agents to improve human health.