Affinity Controls Polyelectrolyte Pair Strength in Complex Coacervate: A Thermodynamic Study

The process of liquid-liquid phase separation plays a fundamental role in organizing different biochemical reactions within intracellular compartments and can give rise to hierarchical compartmentalization, known as membraneless organelles, where multiple immiscible condensates coexist. Complex coacervation, a specific type of associative liquid-liquid phase separation (LLPS) phenomenon, shares many similarities with biomolecular condensates. The strength of interactions governing phase coacervation and the formation of multiphase coacervates is often evaluated based on their resistance to salt-induced dissolution. In this study, we introduce an alternative framework for assessing coacervate immiscibility by examining the binding constant between oppositely charged polyelectrolytes, determined through isothermal titration calorimetry (ITC). By synthesizing peptides with diverse sizes, sequences, and compositions, and utilizing a variety of synthetic and biologically relevant charged species, we gain insight into the interaction affinities between oppositely charged polyelectrolytes. Our results reveal a direct correlation between binding constants and interaction strength, which governs the formation of multiphase coacervates. Additionally, the salt resistance of each polyelectrolyte complex is closely linked to its binding constant. These findings offer valuable insights into the miscibility in polyelectrolyte complex, enhancing our understanding of cellular organization.