Engineering protein and polyelectrolyte complexation for cellular applications

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 polyampholytes, have low charge density, and frequently adopt a globular folded structure. These differences impact the complexation and phase separation of proteins with 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. Additionally, it has recently been demonstrated that the phase separation of proteins is a fundamental mechanism for eukaryotic cellular compartmentalization. These phase separated membraneless organelles create distinct environments that are essential to cellular processes ranging from cell signaling to gene expression. Many membraneless organelles appear to have the same physical properties as complex coacervates – liquid-liquid phase separated mixtures of oppositely charged polyions. We are motivated to understand protein complex coacervation in order to enable new biological applications of these materials. Toward this end, we have investigated the complex coacervation of engineered proteins with synthetic and biological polyions to determine predictive design rules for protein phase separation. We have also used these design rules to promote phase separation of engineered proteins in vivo.