(716h) Sequence and Structure Effects in the Complex Coacervation of Proteins with Polyions

Oppositely charged polyelectrolytes are known to undergo a liquid-liquid phase separation, termed complex coacervation, under the appropriate solution conditions. Protein polyelectrolytes have also been shown to phase separate with polyelectrolytes. However, protein polymers differ significantly from synthetic polyelectrolytes. Proteins are zwitterionic, have low charge density, and typically adopt a globular folded structure. These differences impact the complex coacervation of proteins and polyelectrolytes. These differences also make protein polymers interesting to study in this context; the charge, charge density, and charge orientation on proteins can be precisely controlled through genetic engineering.

Using green fluorescent protein (GFP) as a model protein, the impact of charge density and anisotropy on globular protein phase separation was investigated. Supercharged variants of GFP were biosynthesized with sequence defined charge localized to the protein terminus. Engineered GFP with random distribution of an equivalent charge on the protein surface was also prepared for comparison. Comparison of isotropic and anisotropic charge distribution on GFP provides insight into the mechanism by which charge localization impacts complex coacervation in these systems. Phase separation of the engineered proteins with strong and weak polyelectrolytes has been investigated by light scattering and optical microscopy. Key differences in the phase behavior were observed based on the orientation of charges on the protein. Finally, design rules and short tag sequences for complex coacervation of globular proteins have been established and should enable future applications of these materials.