Construction of Complete Phase Diagrams for Protein Liquid-Liquid Phase Separation by In Situ Raman...

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Liquid-liquid phase separation (LLPS) is crucial for cellular organization.
• Phase diagrams are essential for understanding LLPS conditions but are difficult to determine for proteins.
• Human γD-crystallin is a model protein for studying LLPS.

Purpose of the Study:

• To develop and validate a method for constructing complete phase diagrams of protein LLPS.
• To quantify the temperature-dependent concentrations of coexisting phases for human γD-crystallin.
• To demonstrate the method's applicability in systems with co-solutes.

Main Methods:

• Combined optical microscopy and in situ Raman spectroscopy.
• Quantified protein concentrations in light and dense phases.
• Constructed a complete temperature-dependent phase diagram.

Main Results:

• Successfully constructed a complete phase diagram for human γD-crystallin.
• Demonstrated temperature-dependent concentration changes in both phases.
• Showcased the method's ability to analyze co-solute partitioning.

Conclusions:

• The combined microscopy and Raman spectroscopy approach is effective for determining complete protein LLPS phase diagrams.
• This method provides quantitative insights into protein phase behavior and co-solute interactions.
• The technique is versatile and applicable to complex biological systems.