Wilson C Blake, Lee Myungwoon, Yau Wai-Ming, Tycko Robert
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.
Biophys J. 2024 Dec 3;123(23):4097-4114. doi: 10.1016/j.bpj.2024.11.001. Epub 2024 Nov 4.
Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature T. 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) T. 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 N,C 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%.
FUS蛋白的内在无序低复杂性结构域（FUS-LC）的溶液在温度T以下会发生液-液相分离（LLPS）。为了研究FUS-LC在均匀（即单相）和相分离状态下局部构象分布是否存在可检测到的差异，我们对在远高于（50°C）或远低于（4°C）T的温度下平衡后在亚毫秒时间尺度上冷冻的溶液进行了固态核磁共振（ssNMR）测量。测量在25K下进行，并利用动态核极化增强信号。对于FUS-LC均匀标记N、C的快速冷冻溶液，二维ssNMR谱中的交叉峰模式在两种状态下几乎相同。对于仅在苏氨酸、酪氨酸和甘氨酸残基处标记的FUS-LC溶液，以及通过合成片段与重组片段连接标记五个特定残基的FUS构建体溶液，也获得了类似的结果。这些实验表明，尽管在相分离的富含蛋白质的“液滴”中蛋白质浓度更高且分子间相互作用更丰富，但均匀溶液和相分离溶液中的局部构象分布几乎相同。将实验结果与二维ssNMR交叉峰对β链样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%。
