Biomolecular condensation using de novo designed globular proteins

Abstract

De novo protein design is advancing rapidly, but many targets remain inaccessible to current AI-based tools. Here we describe de novo designed globular domains that drive biomolecular condensation. Starting from a water-soluble, monomeric protein, we make variants with the same amino-acid composition but different surface-charge distributions: one with large patches of surface charge, and another with a more-homogeneous charge distribution. The individual domains form stable and discrete structures in solution, with the large-patch variant exhibiting more-attractive interprotein interactions. Next, two copies of each variant are joined with disordered linkers to generate dumbbell-like proteins. When expressed in eukaryotic cells, the large-patch variant forms intracellular puncta, whereas that with small patches does not. The assemblies are dynamic, liquid condensates in vitro and in cells. The structured domains facilitate functionalisation: we introduce fluorophore-binding sites to visualise fluorescent condensates directly in cells without a GFP reporter; and we manipulate the condensates using motor proteins.