Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature TLLPS. To investigate whether local conformational distributions are detectably different in the homogeneous (i.e., single-phase) and phase-separated states of FUS-LC, we performed solid-state NMR (ssNMR) measurements on solutions that were frozen on submillisecond timescales after equilibration at temperatures well above (50°C) or well below (4°C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich “droplets.” Comparison of the experimental results with simulations of the sensitivity of two-dimensional ssNMR crosspeaks to changes in populations of β strand-like conformations suggests that changes in conformational distributions are no larger than 5–10%.

中文翻译：

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低复杂性蛋白质在均相和相分离冷冻溶液中的构象


FUS 蛋白固有无序、低复杂性结构域 （FUS-LC） 的溶液在低于 TLLPS 温度的温度下进行液-液相分离 （LLPS）。为了研究在 FUS-LC 的均相（即单相）和相分离状态下是否可检测到局部构象分布不同，我们对在远高于 （50°C） 或远低于 （4°C） TLLPS 的温度下平衡后以亚毫秒时间尺度冻结的溶液进行了固体 NMR （ssNMR） 测量。在 25 K 下进行测量，动态核极化的信号增强。发现 FUS-LC 均匀被 15N，13C 标记的快速冷冻溶液的二维 ssNMR 谱图中的交叉峰模式在两种状态下几乎相同。FUS-LC 仅在 Thr、Tyr 和 Gly 残基处标记的溶液，以及通过合成和重组片段连接标记 5 个特异性残基的 FUS 构建体溶液也获得了类似的结果。这些实验表明，尽管相分离、富含蛋白质的“液滴”中的蛋白质浓度要高得多且分子间相互作用更丰富，但在均相和相分离溶液中，局部构象分布几乎相同。将实验结果与二维 ssNMR 交叉峰对 β 链状构象群体变化的敏感性模拟进行比较，表明构象分布的变化不超过 5-10%。