Development of functional synthetic biomolecular condensates.

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PROJECT SUMMARY Membraneless organelles, or biomolecular condensates, have emerged as a strategy to organize the contents of prokaryotic and eukaryotic cells. These phase separated compartments play key roles in a range of cellular functions – from signaling to tuning metabolic pathways or controlling gene expression – yet there are still questions about the fundamental mechanisms for their formation, dynamics, and function. The relationship between condensate molecular properties and function is not yet understood, but this could provide an avenue to treat diseases that involve dysregulated protein condensates (neurodegeneration, cataracts, cancer). Our research proposes to develop functional synthetic biomolecular condensates in order to address several over- arching questions: How do specific intermolecular interactions (electrostatic, cation-π, etc.) contribute to protein phase transitions? How do protein sequence and structure influence the physical properties and function of the condensed phase? Is there a connection between the materials properties and the function of biomolecular condensates? Semi-synthetic biomolecular condensates will allow us to evaluate how molecular interactions in the condensed phase contribute not only to the dynamics of the phase but also to small and macromolecule partitioning, and ultimately the function of endogenous biomolecular condensates. The goals of the proposed research program are to create enzymatically active synthetic membraneless organelles in vitro and in vivo. New materials with varied chemical environments will be prepared and new methods for imaging protein condensates at the molecular scale will be established. These advances will help us to understand how protein sequence influences function at both the microscale (e.g. of an individual enzyme) and the mesoscale (e.g. of a condensed phase cellular compartment). Engineering orthogonal biomolecular condensates has the potential to impact our understanding of the function of native biomolecular condensates and provide a synthetic biology platform to artificially regulate information flow in the cell.