Topology-Encoded Charge Polarity Governs Multiphase Organization in Intrinsically Disordered Protein Polymer...

Synthetic biomolecular condensates offer a route to engineer compartmentalized microenvironments with tunable physicochemical properties, yet design principles for controlling their internal organization remain limited. Here, we report a minimal two-component system based on thermoresponsive intrinsically disordered protein polymers with lower critical solution temperature behavior (LCST-IDPPs), in which electrostatic topology programs liquid-liquid phase separation (LLPS). Incorporating charged residues into LCST-IDPPs suppresses phase separation under physiological conditions, whereas mixing oppositely charged, individually non-coacervating IDPPs restores LLPS as an emergent, composition-dependent process driven by multivalent intermolecular charge compensation. We compare this two-component system with a covalently linked diblock containing the same charged chains. This change in chain connectivity alters the coupling between electrostatic pairing, counterion redistribution, and LCST-driven dehydration. As a result, the balance between inter- and intrachain ionic pairing encodes the residual charge and micropolarity of the dense phase. This topology-dependent microenvironment controls condensate miscibility and drives the formation of either homogeneous or internally demixed multiphase assemblies. The condensate interior also shifts the apparent pK_a of ionizable residues, indicating that phase separation modifies local acid-base equilibria and alters the effective side-chain charge. Together, these findings show how electrostatic topology influences LLPS, the dense-phase microenvironment, and mesoscale organization in IDPP condensates.