The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that formcondensates under physiological conditions often contain intrinsically disordered regions with low-complexitydomains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 mM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in handwith enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation. SIGNIFICANCE Recently, membraneless compartmentalization via phase separation in living cells has been linked to the formation of pathological protein aggregates found in neurodegenerative diseases. Despite its significance, less is known about how liquid-liquid phase separation is triggered or prevented on a molecular scale. Two thermodynamic driving forces have been proposed: protein-protein and protein-water interactions (mostly enthalpic), as well as the release of preorganized hydration water into the bulk (mostly entropic). Whereasmost studies focus on the first aspect, experimental evidence for the latterwas lacking. Here, wepresent a first, toour knowledge, experimental study usinga combination of terahertz spectroscopy and fluorescence microscopy, which reveals that both driving forces are of equal importance for biomolecule condensation.