Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics.

Many multidomain proteins contain disordered linkers that regulate interdomain contacts, and thus the effective concentrations that govern intramolecular reactions. Effective concentrations are rarely measured experimentally, and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction, likely through π-interactions. Finally, we find that the individual contributors to chain compaction are not additive. We thus demonstrate that direct measurement of effective concentrations can be used in systematic studies of the relationship between sequence and structure of intrinsically disordered proteins. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.