期刊
NATURE CHEMICAL BIOLOGY
卷 17, 期 5, 页码 608-614出版社
NATURE PORTFOLIO
DOI: 10.1038/s41589-021-00752-3
关键词
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资金
- Swiss National Science Foundation
- Sinergia [CR-SII5_170976]
- NCCR RNA Disease
- EMBO long-term postdoctoral fellowship
- Volkswagen Foundation Experiment! grant [95664]
The study demonstrates the stabilization of liquid droplets of FUS protein using agarose hydrogel for spectroscopic characterization. The agarose hydrogel acts as a cytoskeleton mimic and cryoprotectant, enabling precise quantification of protein signals in both dispersed and condensed phases, even at cryogenic temperatures. Surprisingly, compact FUS protein was observed in the condensed phase using double electron-electron resonance measurements.
Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.
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