Reversible Disulfide Bond Cross-Links as Tunable Levers of Phase Separation in Designer Biomolecular Condensates

Biomolecular condensates (BCs) are membraneless hubs enriched with proteins and nucleic acids that have emerged as important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is essential in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the past decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to uncover the involvement of disulfide cross-links 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 cross-links, 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 function neither as stickers nor spacers but as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the possible role of cysteines in the formation of biomolecular condensates and their potential use as tunable covalent cross-linkers in developing redox-sensitive viscoelastic materials.