Disulfide Crosslink Density is a Key Determinant in the Formation, Dynamics, and Maturation of Cysteine-rich Designer Biomolecular Condensates.

Biomolecular condensates form through weak, multivalent, non-covalent interactions, but the role of covalent crosslinks in shaping their assembly and material properties remains poorly understood. We previously showed that disulfide bonds between cysteines in designer phase-separating peptides (PSPs) act as redox-reversible crosslinks that lower the saturation concentration (Csat) and help maintain condensate fluidity. Here, we ask which sequence features govern this behavior. We built two peptide libraries: one varying the inter-cysteine spacer length (2-10 residues) and one varying cysteine density (0-33%). Inter-cysteine spacers tuned condensate properties only modestly. Longer inter-cysteine lengths raised Csat from 300 to 900 μM, showed greater fluidity (as assessed by fluorescence recovery after photobleaching (FRAP) recovery rate, and resisted loss of fluidity during maturation; all inter-cysteine length variants dissolved and reformed reversibly over three redox cycles. Cysteine density, in contrast, was the dominant control parameter. Increasing cysteine density lowered Csat about 60-fold (1.5 mM to 25 μM) and reduced the fluidity by an order of magnitude. Its effect on fluidity was non-monotonic: condensates with up to ~14% cysteine density stayed liquid-like for 10 days, whereas those with 21-33% cysteine density in the peptide showed slower coalescence. Condensates with the highest densities also resisted dissolution under reducing conditions and showed sharply reduced uptake of a protein cargo (eGFP; ~60% to ~9%) and a small molecule (TMR, ~70% to ~45%), consistent with smaller mesh size. Together, these results identify disulfide crosslink density as a key lever for programming condensate assembly, viscoelasticity, redox responsiveness, and porosity. The results offer key design principles both for understanding cysteine-rich disordered proteins in biomolecular condensates and for engineering redox-responsive biomaterials.