Phase Separation Behavior of Supercharged Proteins and Polyelectrolytes

Membraneless organelles, like membrane-bound organelles, are essential to cell homeostasis and provide discrete cellular subcompartments. Unlike classical organelles, membraneless organelles possess no physical barrier but rather arise by phase separation of the organelle components from the surrounding cytoplasm or nucleoplasm. Complex coacervation, the liquid-liquid phase separation of oppositely charged polyelectrolytes, is one of several phenomena that are hypothesized to drive the formation and regulation of some membraneless organelles. Studies of the molecular properties of globular proteins that drive complex coacervation are limited as many proteins do not form complexes with oppositely charged macromolecules at neutral pH and moderate ionic strengths. Protein supercharging overcomes this problem and drives complexation with oppositely charged macromolecules. In this work, several distinct cationic supercharged green fluorescent protein (GFP) variants were designed to examine the phase behavior with oppositely charged polyanionic macromolecules. Cationic GFP variants phase separated with oppositely charged macromolecules at various mixing ratios, salt concentrations, and pH values. Efficient protein incorporation in the macromolecule rich phase occurred over a range of protein and polymer mass fractions, but the protein encapsulation efficiency was highest at the midpoint of the phase separation regime. More positively charged proteins phase separated over broader pH and salt ranges than those of proteins with a lower charge density. Interestingly, each GFP variant phase separated at higher salt concentrations with anionic synthetic macromolecules compared to anionic biological macromolecules. Optical microscopy revealed that most variants, depending on solution conditions, formed liquid-liquid phase separations, except for GFP/DNA pairs that formed solid aggregates under all tested conditions.