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

Area of Science:

• Biomolecular Engineering
• Soft Matter Physics
• Polymer Science

Background:

• Synthetic biomolecular condensates allow engineering of compartmentalized microenvironments.
• Controlling the internal organization of these condensates remains a challenge.
• Intrinsically disordered protein polymers (IDPPs) are key components in condensate formation.

Purpose of the Study:

• To investigate how electrostatic topology influences liquid-liquid phase separation (LLPS) in thermoresponsive IDPPs.
• To understand the design principles for controlling the internal organization and physicochemical properties of biomolecular condensates.
• To explore the impact of chain connectivity on LLPS and condensate properties.

Main Methods:

• Development of a minimal two-component system using oppositely charged, thermoresponsive IDPPs.
• Comparison with a covalently linked diblock system.
• Analysis of LLPS behavior, condensate miscibility, and internal microenvironment properties.
• Investigation of the effect of phase separation on acid-base equilibria within condensates.

Main Results:

• Mixing oppositely charged, non-coacervating IDPPs restored LLPS as a composition-dependent process driven by charge compensation.
• Chain connectivity (two-component vs. diblock) altered the coupling between electrostatics, counterion redistribution, and dehydration, affecting condensate properties.
• Electrostatic topology determined the residual charge and micropolarity of the dense phase, controlling condensate miscibility and leading to homogeneous or multiphase assemblies.
• Phase separation shifted the apparent pKa of ionizable residues, modifying local acid-base equilibria.

Conclusions:

• Electrostatic topology is a critical design principle for programming LLPS and controlling the mesoscale organization of IDPP condensates.
• The internal microenvironment of condensates, including their micropolarity and acid-base properties, is tunable via electrostatic interactions and chain connectivity.
• This work provides a framework for engineering synthetic condensates with tailored internal organization and physicochemical properties.