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 formcondensatesunderphysiologicalconditionsoftencontainintrinsicallydisorderedregionswithlow-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 de-mentia. 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 proposedaspect isthe release ofentropically unfavorable preorderedhydrationwaterinto the bulk. Weusedattenuatedtotal reflection spectroscopy in theterahertz frequency range to characterize thechangesin the hydrogen bondingnetwork 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 m M 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 thebulkgoeshandinhandwithenthalpicallyfavorableprotein-proteininteraction.Bothchangesareenergeticallyfavorable,andour study shows that both contribute to the thermodynamic driving force in 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). Whereas most studies focus on the first aspect, experimental evidence for the (cid:5)

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