Liquid–Liquid Phase Separation? Ask the Water!

Water is more than an inert spectator during liquid-liquid phase separation (LLPS), the reversible compartmentalization of protein solutions into a protein-rich and a dilute phase. We show that LLPS is driven by changes in hydration entropy and enthalpy. Tuning LLPS by adjusting experimental parameters, e.g., addition of co-solutes, is a major goal for biological and medical applications. This requires a general model to quantify thermodynamic driving forces. Here, we develop such a model based on the measured amplitudes of characteristic THz-features of two hydration populations: "Cavity-wrap" water hydrating hydrophobic patches is released during LLPS leading to an increase in entropy. "Bound" water hydrating hydrophilic patches is retained since it is enthalpically favorable. We introduce a THz-phase diagram mapping these spectroscopic/thermodynamic changes. This provides not only a precise understanding of hydrophobic and hydrophilic hydration driving forces as a function of temperature and concentration but also a rational means to tune LLPS.