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

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. (c) 2025 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.