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 20892.
current address: Department of Chemistry, Drexel University, Philadelphia, PA 19104.
bioRxiv. 2024 Jul 25:2024.07.25.605144. doi: 10.1101/2024.07.25.605144.
Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below temperatures T in the 20-40° C range. To investigate whether local conformational distributions are detectably different in the homogeneous and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales 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 (2D) 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 2D crosspeak patterns to an enhanced population of β-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%.
Liquid-liquid phase separation (LLPS) in solutions of proteins with intrinsically disordered domains has attracted recent attention because of its relevance to multiple biological processes and its inherent interest from the standpoint of protein biophysics. The high protein concentrations and abundant intermolecular interactions within protein-rich, phase-separated "droplets" suggests that conformational distributions of intrinsically disordered proteins may differ in homogeneous and phase-separated solutions. To investigate whether detectable differences exist, we performed experiments on the low-complexity domain of the FUS protein (FUS-LC) in which FUS-LC solutions were first equilibrated at temperatures well above or well below their LLPS transition temperatures, then rapidly frozen and examined at very low temperatures by solid state nuclear magnetic resonance (ssNMR) spectroscopy. The ssNMR data for homogeneous and phase-separated frozen solutions of FUS-LC were found to be nearly identical, showing that LLPS is not accompanied by substantial changes in the local conformational distributions of this intrinsically disordered protein.
FUS蛋白的内在无序低复杂性结构域（FUS-LC）的溶液在20-40°C范围内的温度T以下会发生液-液相分离（LLPS）。为了研究FUS-LC在均匀态和相分离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，利用动态核极化增强信号。在FUS-LC均匀N、C标记的快速冷冻溶液的二维（2D）ssNMR谱中，发现两种状态的交叉峰模式几乎相同。对于仅在苏氨酸、酪氨酸和甘氨酸残基处标记的FUS-LC溶液，以及通过合成片段和重组片段连接对五个特定残基进行标记的FUS构建体溶液，也获得了类似结果。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。将实验结果与二维交叉峰模式对β-链样构象增强群体敏感性的模拟结果进行比较表明，构象分布的变化不超过5-10%。
具有内在无序结构域的蛋白质溶液中的液-液相分离（LLPS）因其与多种生物过程的相关性以及从蛋白质生物物理学角度来看的内在趣味性，最近受到了关注。富含蛋白质的相分离“液滴”中高蛋白质浓度和丰富的分子间相互作用表明，内在无序蛋白质的构象分布在均匀溶液和相分离溶液中可能不同。为了研究是否存在可检测到的差异，我们对FUS蛋白的低复杂性结构域（FUS-LC）进行了实验，其中FUS-LC溶液首先在远高于或远低于其LLPS转变温度的温度下平衡，然后快速冷冻，并在非常低的温度下通过固态核磁共振（ssNMR）光谱进行检测。发现FUS-LC均匀冷冻溶液和相分离冷冻溶液的ssNMR数据几乎相同，表明LLPS不会伴随这种内在无序蛋白质局部构象分布的实质性变化。
