Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid–liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely β conformations, analogous to models of pathological phase separation.