Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Many cellular proteins have the ability to demix spontaneously from solution to form liquid condensates. These phase-separated structures form membraneless compartments in living cells and have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid-liquid phase separation (LLPS) of proteins has thus become a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular phase separation, such as FUS, TDP-43, and Annexin A11, which form condensates at low salt concentrations via homotypic multivalent interactions, also have the ability to undergo LLPS at high salt concentrations by reentering into a phase-separated regime. Through a combination of experiments and simulations, we demonstrate that phase separation in the high-salt regime is mainly driven by hydrophobic and non-ionic interactions. As such, it is mechanistically distinct from the low-salt regime, where condensates are stabilized by a broad mix of electrostatic, hydrophobic, and non-ionic forces. Our work thus expands the molecular grammar of interactions governing LLPS of cellular proteins and provides a new view on hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for the aberrant function, druggability, and material properties of biomolecular condensates.