Sequence grammar underlying unfolding and phase separation of globular proteins

Abstract

Protein homeostasis involves regulation of the concentrations of unfolded states of globular proteins. Dysregulation can cause phase separation leading to protein-rich deposits. Here, we uncover the sequence-grammar that influences the triad of folding, binding, and phase equilibria of unfolded proteins in cells. We find that the interactions that drive deposit formation of ALS-associated superoxide dismutase 1 mutations are akin to those that drive phase separation and deposit formation in variants of a model protein, barnase. We examined a set of barnase variants to uncover the molecular interactions that drive phase separation of unfolded proteins and formation of unfolded protein deposits (UPODs). The formation of UPODs requires protein destabilization, to increase the concentration of unfolded states, and a requisite sequence grammar to enable cohesive interactions among unfolded proteins. We further find that molecular chaperones, Hsp40 and Hsp70, destabilize UPODs by binding preferentially to and processing unfolded proteins in the dilute phase. Highlights Unfolded states of globular proteins undergo aggregation-mediated phase separation to form unfolded protein deposits (UPODs) in cells. The threshold concentration of unfolded proteins required to drive phase separation is governed by a combination of protein stability and the requisite grammar of cohesive motifs known as stickers that provide cohesive intermolecular interactions. The sequence grammar that contributes to phase separation of unfolded proteins shares features with those of proteins that drive the formation of functional biomolecular condensates. Molecular chaperones regulate the concentrations of free unfolded proteins in cells by binding preferentially to unfolded states in dilute phases thereby destabilizing UPOD formation mainly via binding-mediated polyphasic linkage.