Investigating Different Dynamic pHP1α States in Their KCl-Mediated Liquid-Liquid Phase Separation (LLPS) Using...

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Chromatin phase separation is crucial for genomic activities and is regulated by various factors.
• Liquid-liquid phase separation (LLPS) of chromatin and proteins is observed in vitro and in vivo, but mechanisms remain unclear.
• Physicochemical properties of phase-separated complexes are challenging to study.

Purpose of the Study:

• To investigate the dynamic and physicochemical properties of phosphorylated heterochromatin protein 1α (pHP1α) phase-separated systems.
• To elucidate the role of molecular motion and salt concentration in regulating chromatin phase separation.

Main Methods:

• Utilized modified solid-state NMR (SSNMR) pulse sequences, including heteronuclear Overhauser effect (hetNOE), to detect dynamic components.
• Employed NMR techniques to selectively excite and study signals from different states within the protein gel.
• Integrated molecular dynamics (MD) simulations with experimental NMR data.

Main Results:

• Detected dynamic, viscous, and intermediate components in the pHP1α phase-separated system.
• Observed reduced molecular motion at low KCl concentration (30 mM).
• Found increased molecular motion at high KCl concentration (150 mM), attributed to electrostatic interactions.

Conclusions:

• Modified SSNMR, particularly hetNOE, provides direct insight into molecular dynamics of phase-separated systems.
• KCl concentration significantly influences molecular motion in pHP1α phase separation.
• Intermolecular electrostatic interactions, modulated by KCl, play a key role in regulating the dynamics of chromatin phase separation.