Phase Separation Behavior of Supercharged Proteins and Polyelectrolytes.

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Membraneless organelles are crucial for cellular homeostasis and function without physical barriers.
• Complex coacervation, a type of liquid-liquid phase separation, is a proposed mechanism for membraneless organelle formation.
• Studying protein-driven complex coacervation is challenging due to limited protein complexation at neutral conditions.

Purpose of the Study:

• To investigate the phase behavior of supercharged cationic green fluorescent protein (GFP) variants with anionic macromolecules.
• To understand how protein charge density influences complex coacervation and membraneless organelle formation.
• To explore the impact of salt concentration, pH, and macromolecule type on protein-polymer phase separation.

Main Methods:

• Designing and synthesizing several distinct cationic supercharged GFP variants.
• Inducing complex coacervation by mixing supercharged GFP variants with oppositely charged polyanionic macromolecules.
• Analyzing phase separation behavior across varying mixing ratios, salt concentrations, and pH values.
• Utilizing optical microscopy to characterize the resulting phase separated structures.

Main Results:

• Cationic GFP variants successfully underwent phase separation with anionic macromolecules under diverse conditions.
• Optimal protein incorporation into the macromolecule-rich phase was observed at the midpoint of the phase separation regime.
• Higher protein charge density correlated with broader pH and salt concentration ranges for phase separation.
• Supercharged proteins showed distinct phase separation behaviors with synthetic versus biological anionic macromolecules, with DNA forming solid aggregates.

Conclusions:

• Protein supercharging is an effective strategy to drive complex coacervation and study membraneless organelle formation.
• Protein charge density significantly modulates the conditions required for phase separation.
• The type of anionic macromolecule influences the phase behavior, with implications for understanding in vivo organelle formation.