Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy.
Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy.
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DOI：
10.1038/s41467-022-29408-z
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发表时间：
2022-04-01
影响因子：
16.6
通讯作者：
Golovanov AP
中科院分区：
文献类型：
--
作者：
Bramham JE;Golovanov AP
蛋白质溶液的液-液相分离（LLPS）是细胞生物学和生物技术中的一个重要现象。然而，乳白色和浓度波动使得LLPS难以研究，特别是在表征相变和层分离动力学时。在这里，我们展示了使用探针分子三氟乙醇（TFE）通过核磁共振光谱表征蛋白质LLPS的动力学。探针分子的化学位移和线宽对局部蛋白质浓度敏感，这种敏感性导致稠密相和稀薄相产生不同的特征信号。通过传统的批量检测19F核磁共振监测这些探针信号，报告了整个样品中两相的形成和演变，包括它们的浓度和体积。同时，利用空间选择性19F NMR（从较小的样品切片记录光谱）来跟踪层分离过程中不同相的分布。该实验策略能够全面表征LLPS的过程和动力学，并可能有助于研究蛋白质系统中相分离作为其环境的函数。蛋白质液-液相分离是生物学中的重要现象。在这里，作者展示了一种在核磁共振光谱中使用氟化探针分子表征蛋白质相在时间和空间上的演变的方法。
Liquid-liquid phase separation (LLPS) of protein solutions is increasingly recognised as an important phenomenon in cell biology and biotechnology. However, opalescence and concentration fluctuations render LLPS difficult to study, particularly when characterising the kinetics of the phase transition and layer separation. Here, we demonstrate the use of a probe molecule trifluoroethanol (TFE) to characterise the kinetics of protein LLPS by NMR spectroscopy. The chemical shift and linewidth of the probe molecule are sensitive to local protein concentration, with this sensitivity resulting in different characteristic signals arising from the dense and lean phases. Monitoring of these probe signals by conventional bulk-detection 19F NMR reports on the formation and evolution of both phases throughout the sample, including their concentrations and volumes. Meanwhile, spatially-selective 19F NMR, in which spectra are recorded from smaller slices of the sample, was used to track the distribution of the different phases during layer separation. This experimental strategy enables comprehensive characterisation of the process and kinetics of LLPS, and may be useful to study phase separation in protein systems as a function of their environment. Protein liquid-liquid phase separation is an important phenomenon in biology. Here, the authors demonstrate an approach to characterize the evolution of protein phases in both time and space using a fluorinated probe molecule in NMR spectroscopy.
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影响因子：
5.7
作者：
Bucciarelli, Saskia;Mahmoudi, Najet;Stradner, Anna
通讯作者：
Stradner, Anna
影响因子：
2.9
作者：
LEE J C;TIMASHEFF S N
通讯作者：
TIMASHEFF S N
DOI：
10.1073/pnas.110000497
发表时间：
2000-06-06
影响因子：
11.1
作者：
Galkin, O;Vekilov, PG
通讯作者：
Vekilov, PG
影响因子：
16.6
作者：
Ambadipudi S;Biernat J;Riedel D;Mandelkow E;Zweckstetter M
通讯作者：
Zweckstetter M
DOI：
10.1073/pnas.1706197114
发表时间：
2017-09-26
影响因子：
11.1
作者：
Brady, Jacob P.;Farber, Patrick J.;Kay, Lewis E.
通讯作者：
Kay, Lewis E.