Globular proteins can undergo a liquid-liquid phase separation, termed complex coacervation, with oppositely charged polyelectrolytes. This macromolecule rich liquid phase can be used to stabilize the protein component, mimic the cytoplasmic environment, or enhance protein activity. However, due to the relatively low charge density and “patchy” charged surface of most proteins, many globular proteins do not phase separate with oppositely charged polyelectrolytes. Efforts to understand the parameters that govern globular protein complex coacervation have focused on commercially available proteins and chemically modified variants of these proteins. These studies have revealed the importance of the ratio of charged residues on the protein and localized “charge patches.” In order to develop design criteria and engineering strategies for globular protein complex coacervation, additional understanding of the factors that control protein-polyelectrolyte phase separation is needed.<br/><br/>Genetic modification of recombinantly expressed proteins enables the study of protein parameters that influence complex coacervation. Using green fluorescent protein (GFP) as a model protein, the impact of charge anisotropy on globular protein phase separation was investigated. Supercharged variants of GFP were biosynthesized with random distribution of charge on the protein surface. The net charge on the protein surface was varied from +30 to -30. Concurrently, the wild type GFP (with a net charge of -7) was modified at the C-terminus to vary the overall charge from +30 to -30 and provide an anisotropic, localized charge patch. Comparison of isotropic and anisotropic charge distribution on GFP provides insight into the mechanism by which charge localization impacts complex coacervation in these systems.

Globular protein based complex coacervates

254th National Meeting (2017)

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