Intrinsically disordered proteins (IDPs) that undergo liquid-liquid phase separation (LLPS) play a vital role in the formation of membraneless organelles, which are essential for numerous cellular functions and regulatory processes, yet may also contribute to diseases of protein aggregation. The amino acid sequence of IDPs governs their inter- and intra-molecular interactions, propensity for phase separation, and material properties of their condensates. Previously, we identified key sequence features that contribute to phase separation of engineered IDPs; the condensates formed were viscous and not prone to aggregation. Here, we asked what mutations would cause the engineered IDPs to rapidly age from liquid to other material states. We generated sequence variants that retained the ability to undergo LLPS, with vastly different propensity for phase separation. Notably, several sequences exhibited dramatic time-dependent changes to their condensate material properties, including liquid-to-gel or liquid-to-solid transition, and even fibril formation, depending on sequence composition. To conclude, these findings underscore the importance and complexity of protein sequence in modulating both phase behavior and physical properties of condensates. This study can contribute to the further understanding of sequence determinants underlying biomolecular condensation and aggregation and provide valuable insights for engineering programmable synthetic IDPs for synthetic biology and biomaterials applications.
