A combined approach to extract rotational dynamics of globular proteins undergoing liquid-liquid phase separation

Dominik Gendreizig, Abhishek Kalarikkal, Simon L. Holtbrügge, Saumyak Mukherjee, Laura Galazzo, Svetlana Kucher, Arnulf Rosspeintner, Lars V. Schäfer, Enrica Bordignon
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Abstract

The formation of protein condensates (droplets) via liquid-liquid phase separation (LLPS) is a commonly observed phenomenon in vitro. Changing the environmental properties with cosolutes, molecular crowders, protein partners, temperature, pressure, etc. was shown to favour or disfavour the formation of protein droplets by fine-tuning the water-water, water-protein and protein-protein interactions. Therefore, these environmental properties and their spatiotemporal fine-tuning are likely to be important also in a cellular context at the existing protein expression levels. One of the key physicochemical properties of biomolecules impacted by molecular crowding is diffusion, which determines the viscoelastic behaviour of the condensates. Here we investigate the change in the rotational diffusion of γD-crystallin, undergoing LLPS in vitro in aqueous solutions in absence and presence of cosolutes. We studied its rotational dynamics using molecular dynamics simulations (MD), electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy. MD simulations performed under dilute and crowded conditions show that the rotational diffusion of crystallin in water is retarded by one to two orders of magnitude in the condensed phase. To obtain the rotational dynamics in the dilute phase we used fluorescence anisotropy and to extract the retardation factor in in the condensed phase we used spin-labeled γD-crystallin proteins as EPR viscosity nanoprobes. Aided by a viscosity nanoruler calibrated with solutions at increasing sucrose concentrations, we validate the rotational diffusion retardation predicted by MD simulations. This study underlines the predictive power of MD simulations and showcases the use of a sensitive EPR nanoprobe to extract the viscosity of biomolecular condensates.
提取发生液-液相分离的球状蛋白质旋转动力学的组合方法
通过液-液相分离(LLPS)形成蛋白质凝聚物(液滴)是体外观察到的常见现象。通过微调水-水、水-蛋白质和蛋白质-蛋白质之间的相互作用,改变共溶质、分子排阻剂、蛋白质伙伴、温度、压力等环境属性,有利于或不利于蛋白质液滴的形成。因此,这些环境特性及其时空微调很可能在现有蛋白质表达水平的细胞环境中也很重要。扩散是受分子拥挤影响的生物大分子的关键物理化学特性之一,它决定了凝聚物的粘弹性行为。在此,我们研究了γ-D-结晶素在体外水溶液中,在没有共溶质和有共溶质的情况下进行 LLPS 时旋转扩散的变化。我们利用分子动力学模拟(MD)、电子顺磁共振(EPR)光谱和荧光光谱对其旋转动力学进行了研究。在稀释和拥挤条件下进行的 MD 模拟显示,结晶素在水中的旋转扩散在凝聚相中会被延缓一到两个数量级。为了获得稀释相中的旋转动力学,我们使用了荧光各向异性;为了提取凝聚相中的延迟因子,我们使用了自旋标记的γD-结晶蛋白作为 EPR 粘度纳米探针。在用蔗糖浓度不断增加的溶液校准的粘度纳米标尺的帮助下,我们验证了 MD 模拟预测的旋转扩散延迟。这项研究强调了 MD 模拟的预测能力,并展示了使用灵敏的 EPR 纳米探针提取生物分子凝聚物粘度的方法。
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