Two‐ and Three‐Hybrid Systems

Abstract

One goal of chemical biology is to document and understand the macromolecular interactions that take place in the cell. In this review, we discuss the classic yeast two‐hybrid method (and its derivatives) and how this method continues to provide insight into the number of protein–protein partnerships in a cell. In turn, these platforms have been adapted to explore a variety of interactions, such as those between proteins and small molecules. These systems collectively are called three‐hybrid assays, and they provide new opportunities for the discovery of potential binding pairs. However, two‐ and three‐hybrid assays also have significant disadvantages, such as incomplete coverage and high rates of false positives. With these issues in mind, we discuss emerging ways of minimizing the impact of these limitations using microarrays and mass spectrometry. Finally, chemical probes related to the three‐hybrid concept are going beyond observation and providing active, rational control over individual protein–protein contacts. In these systems, bifunctional compounds are used to homo‐ or heterodimerize target proteins reversibly, thus altering their colocalization. By purposefully controlling protein–protein contacts, these chemical dimerization methods have progressed beyond observation and towards synthetic manipulation of protein function.