Hilditch, A. T. | Search bioRxiv

bioRxiv2024

Maturation and conformational switching of a de novo designed phase-separating polypeptide

Cellular compartments formed by biomolecular condensation are a widespread feature 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 arise 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 the 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 {beta} conformations, analogous to models of pathological phase separation.

synthetic biology↗

bioRxiv2023

Assembling membraneless organelles from de novo designed proteins

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems. The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge, which also presents opportunities for chemical and synthetic biology. Here, we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli (E. coli). To do this, we combine de novo -helical sequences, intrinsically disordered linkers, and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from diffusion-limited assemblies to dynamic condensates. The designs are characterised in cells and in vitro using biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair in E. coli.

synthetic biology↗