Department Physical Chemistry, Ruhr-University Bochum, Bochum, Germany.
Department Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr-University Bochum, Bochum, Germany.
Biophys J. 2021 Apr 6;120(7):1266-1275. doi: 10.1016/j.bpj.2021.01.019. Epub 2021 Jan 28.
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 form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. 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 μM 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 hand with 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.
生物分子凝聚物通过液-液相分离（LLPS）的形成已经成为细胞生物学中的一个普遍原则，允许对动态细胞过程进行区室化和时空调节。在生理条件下形成凝聚物的蛋白质通常含有低复杂度结构域的固有无序区域。其中，RNA 结合蛋白 FUS 和 TDP-43 一直是研究的焦点，因为这些蛋白质的异常凝聚和聚集与肌萎缩侧索硬化症和额颞叶痴呆等神经退行性疾病有关。当富含蛋白质的凝聚物形成时，LLPS 就会发生，周围是稀水溶液。LLPS 本身从热力学角度来看是不利的。能量有利的多价蛋白质-蛋白质相互作用是抵消熵成本的一个重要方面。另一个被提出的方面是将熵不利的预有序水合进入主体。我们使用太赫兹频域的衰减全反射光谱来描述 FUS 在液-液相分离液滴中富集时伴随的氢键网络的变化，为溶剂作为热力学驱动力的关键作用提供实验证据。通过荧光测量，确定 LLPS 液滴内的 FUS 浓度独立于初始蛋白质浓度（5 或 10 μM 溶液）增加到 2.0 mM。通过太赫兹光谱，我们揭示了分离的 FUS 中疏水性侧链的去湿。因此，熵不利的水群体释放到主体中与焓有利的蛋白质-蛋白质相互作用同时发生。这两种变化在能量上都是有利的，我们的研究表明，这两种变化都有助于相分离中的热力学驱动力。
