(563a) Engineering Electrostatic Interactions between Proteins and Biopolymers for Intracellular Phase Separation

Protein and nucleic acid phase separation is key to several biological processes, from cell signaling to RNA processing. In addition to specific biological interactions between these biomolecules, electrostatic interactions between positively charged protein domains and negatively charged nucleic acids have been shown to play a role in regulating the formation of these intracellular condensed phases. To directly probe the importance of electrostatic interactions in intracellular phase separation, we have engineered a panel of both cationic and anionic proteins and evaluated their phase behavior in simulated in vivo conditions as well as directly inside living cells. In vitro, phase separation between proteins and RNA was evaluated by turbidity measurements and optical microscopy. We also monitored intracellular phase separation in E. coli by fluorescence microscopy. Both in vitro and in bacteria, we have found that supercationic proteins form complex coacervates with RNA and enrich a small fraction of endogenous proteins in the condensed phase. Additionally, we have identified short cationic sequences that are sufficient to drive intracellular complex coacervation of globular proteins with modest net charge. Phase separation can be predicted by the overall net protein charge, regardless of distribution between the globular domain and the short peptide sequence. Building on our findings with model proteins, we have also engineered intracellular condensates that contain enzymes and demonstrated that they maintain activity in the condensed phase.