Citation: Ruotong Wei, Aokun Liu, Jian Kuang, Zhiwen Wang, Lu Yu, Changlin Tian. Probing the dynamic properties in the LLPS process via site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy[J]. Chinese Chemical Letters, ;2025, 36(4): 110029. doi: 10.1016/j.cclet.2024.110029 shu

Probing the dynamic properties in the LLPS process via site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy

Ruotong Wei ^{a, b} ,
Aokun Liu ^{a, b} ,
Jian Kuang ^b ,
Zhiwen Wang ^b ,
Lu Yu ^{a, b, *,} ,
Changlin Tian ^{a, b, *,}

a.
High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China
b.
Department of Endocrinology, Institute of Endocrine and Metabolic Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Joint Center for Biological Analytical Chemistry, Anhui Engineering Laboratory of Peptide Drug, Anhui Laboratory of Advanced Photonic Science and Technology, University of Science and Technology of China, Hefei 230026, China

luyuesr@ustc.edu.cn

cltian@ustc.edu.cn

Received Date: 18 March 2024
Revised Date: 16 May 2024
Accepted Date: 19 May 2024
Available Online: 19 May 2024

Figures(3)

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a common phenomenon in cells that underlies the formation of membraneless organelles. Although the macroscopic behavior of biomolecular coacervates has been elucidated by microscopy, the detailed dynamic properties of proteins/peptides during the LLPS process remain poorly characterized. Here, site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy was employed to characterize the dynamic properties of a minimal model LLPS system consisting of positively charged peptides and RNA. The degree of phase separation, indicated by broadening of the EPR spectrum of the spin-labeled peptide due to slow molecular tumbling, was monitored by EPR. In addition, three distinct populations with varying molecular motion during LLPS, featuring different spectral lineshapes, were identified. These populations included a fast motion component (Ⅰ), a slower motion component (Ⅱ) associated with peptides in the dispersed phase and an immobile component (Ⅲ) observed in the dense phase. With gradual titration of the peptides to RNA, the EPR spectrum gradually shifted, reflecting changes in the populations of the components. Together, SDSL-EPR method not only provides new insights into the dynamic behavior of biomolecules during LLPS, but also offers a sensitive method for biomolecular phase separation processes at the molecular level.
- Site-directed spin labeling (SDSL),
- Electron paramagnetic resonance (EPR),
- Phase separation,
- Dynamic properties,
- Molecular tumbling
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