Protein–protein interactions (PPIs) are the “handshakes” that let proteins assemble into machines, relay signals, build cellular structures, and decide cell fate. Chemical biology approaches PPIs with a distinctive philosophy: instead of only observing interactions, it builds molecules that can measure, perturb, stabilize, or rewire them—often in living systems—so interaction networks become experimentally controllable rather than just describable. This article is a knowledge-oriented deep dive into how Chemical Biology studies PPIs, what the major experimental strategies are, and how to think clearly about interaction “truth” versus experimental artifacts. 1) Why PPIs are hard: the core scientific challenge Many PPIs are not like enzyme–substrate binding (deep pockets and rigid fits). Instead, a large fraction are: Interface-dominated: broad, shallow surfaces rather than a single pocket. Dynamic: transient contacts that appear only at certain times, locations, or cellular states. Context dependent: the same pair of proteins may interact in one cell type but not another, or only after a modification (phosphorylation, ubiquitination, etc.). So PPI science is less about “does A bind B?” and more about: When and where does A approach B? Is it direct binding or complex membership (A and B in the same assembly but not touching)?…
