Affinity Controls Polyelectrolyte Pair Strength in Complex Coacervate: A Titration Calorimetry Investigation

Liquid-liquid phase separation is recognized as a key mechanism for organizing biochemical reactions within distinct intracellular compartments, and potentially hierarchical compartmentalization, where multiple immiscible condensates are present. Complex coacervation is a liquid-liquid phase separation phenomenon with many parallels to biomolecular condensates. The strength of interactions driving phase coacervation, and the potential for forming multiphase coacervates is often characterized with regards to the resistance to dissolution by salt. We propose a novel approach to understanding the potential for coacervate immiscibility based on the binding constant between the oppositely-charged polyelectrolytes, measured via isothermal titration calorimetry. By synthesizing peptides with varying sizes, sequences, and compositions, and employing a range of synthetic and biological charged species, we provide insights into the interaction affinities between oppositely charged polyelectrolytes. Our findings show that differences in binding constants correlate with interaction strength, which explains the formation of multiphase coacervates. Furthermore, the salt resistance of each polyelectrolyte complex is directly related to its binding constant.