Electrostatic interactions drive phase separation in Pup protein

The similarities in the liquid-liquid phase separation (LLPS) phenomenon between membrane-less organelles (MLOs) and coacervates make them an ideal platform for studying the nature of life. Herein, we have developed a peptide-protein coacervative system between the positively charged poly-L-lysine peptide and negatively charged intrinsically disordered Pup protein to model the electrostatic nature of MLOs, providing insights into their in vivo mechanisms and potential therapeutic targets. We have constructed a phase diagram for coacervate formation at different concentrations of the Pup-PLL system, determined by turbidity measurements and optical microscopy. Fluorescence microscopy confirms the presence of both the polymers in the coacervates and the pH dependent studies confirm the role of electrostatics and emphasize the significance of charge density during coacervation.