Assembling membraneless organelles fromde novodesigned proteins

Abstract

Recent advances inde novoprotein design have delivered a diversity of discretede novoprotein 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 inEscherichia coli(E. coli). To do this, we combinede novoα-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 andin vitrousing biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair inE. coli.