Reversible disulfide bond crosslinks as tunable levers of phase separation in designer biomolecular condensates

Biomolecular condensates (BCs) are membraneless hubs enriched in proteins and nucleic acids that have become important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is important in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the last decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to determine the involvement of disulfide crosslinks and their redox sensitivity in BCs, we designed a 'stickers and spacers' model of phase-separating peptides interspersed with cysteines. Through biophysical investigations, we learned that cysteines promote liquid-liquid phase separation in oxidizing conditions and perpetuate liquid condensates through disulfide crosslinks, which can be reversibly tuned with redox chemistry. By varying the composition of cysteines, subtle but distinct changes in the viscoelastic behavior of the condensates were observed. Empirically, we conclude that cysteines are neither stickers nor spacers but function as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the role of cysteines in protein phase behavior and the potential to develop tunable, redox-sensitive viscoelastic materials.