Observation of the Liquid-Liquid Phase Separation of FUS-LC Using Vacuum-Ultraviolet Circular Dichroism Spectroscopy

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• The low-complexity domain of fused in sarcoma protein (FUS-LC) is implicated in liquid-liquid phase separation (LLPS).
• Understanding the structural mechanisms of FUS-LC LLPS is crucial for comprehending its biological roles and potential disease associations.

Purpose of the Study:

• To elucidate the structural basis of FUS-LC-mediated liquid-liquid phase separation (LLPS).
• To investigate the secondary structural changes of FUS-LC during LLPS using advanced spectroscopic techniques.

Main Methods:

• Vacuum-ultraviolet circular dichroism (VUV-CD) spectroscopy was employed to analyze protein secondary structures.
• CD spectra were measured across a temperature range (37°C to 5°C) to induce and observe LLPS of FUS-LC.
• Measurements were conducted at the BL12 VUV-CD station at the Hiroshima Synchrotron Radiation Center (HiSOR).

Main Results:

• The CD spectrum of FUS-LC at 37°C showed a prominent negative peak at 195 nm and a shoulder near 220 nm.
• A decrease in peak intensity around 195 nm was observed as the temperature was lowered.
• These spectral alterations correlated directly with the formation of FUS-LC liquid-liquid phase separation.

Conclusions:

• The study reveals temperature-dependent structural changes in FUS-LC associated with LLPS.
• VUV-CD spectroscopy is effective in characterizing the structural transitions during protein LLPS.
• The findings provide insights into the molecular mechanisms governing FUS-LC phase separation.