Assembly and functionalisation of membraneless organelles from de novo designed proteins

Abstract

In recent years, protein design has undergone a revolution, giving rise to increasingly complex protein assemblies with atomistic accuracy. This has unlocked the potential to design sophisticated macromolecular complexes that can emulate biological systems. As one of the foundations of cellular organisation, the design of artificial organelles is a key target for synthetic biologists. Designed assemblies emulating bacterial microcompartments or fibrous scaffolds have been accessible for some time. A less-explored mechanism for protein assembly is by liquid-liquid phase separation. Despite its newfound relevance to cell biology, the design of protein assemblies that can reversibly de-mix is still in its infancy.

In this thesis, the bottom-up design of proteins for liquid-liquid phase separation is described. These proteins use de novo α-helical coiled coils as protein-protein interaction motifs, combined with unstructured linkers, to drive self-assembly in cells. Using rational coiled coil design principles, the interactions between these motifs are calibrated to drive dynamic liquid-liquid de-mixing, over arrested aggregation. Soft-matter biophysical techniques confirm the liquid-like properties of the de novo assemblies both in vitro and in Escherichia coli (E. coli). Moreover, the designer condensates can be functionalised with catalytic enzymes. Co-condensation of the two enzyme pathway for the production of indigo dye in the de novo organelles results in 6-fold more product than the comparable free enzymes.

In addition to phase separation, the formation of macromolecular fibres by the designed proteins is described. Analogous to droplet maturation, the designed proteins can form both fibres and de-mixed droplets in cells. Finally, following functionalisation in bacteria, phase separation of the designed helical proteins is investigated in mammalian cells. Here, the assemblies are re-designed for phase separation within the larger, and less crowded, eukaryotic cytoplasm. Taken together, these studies begin to highlight the power of bottom-up design for the construction and rationalisation of protein condensates.

Date of Award	3 Oct 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Dek N Woolfson (Supervisor) & Nigel J Savery (Supervisor)