Mondal Malay, Jankoski Penelope E, Lee Landon D, Dinakarapandian Daniel M, Chiu Tzu-Ying, Swetman Windfield S, Wu Hongwei, Paravastu Anant K, Clemons Tristan D, Rangachari Vijayaraghavan
bioRxiv. 2024 Jul 17:2024.07.13.603402. doi: 10.1101/2024.07.13.603402.
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.
生物分子凝聚物（BCs）是富含蛋白质和核酸的无膜中心，已成为许多细胞功能中的重要参与者。揭示蛋白质相分离的序列决定因素对于理解BCs的生物物理和生化特性至关重要。尽管在过去十年中有重大发现，但半胱氨酸残基在BCs形成和溶解中的作用仍然未知。在这里，为了确定二硫键交联及其氧化还原敏感性在BCs中的作用，我们设计了一种穿插有半胱氨酸的相分离肽的“贴纸和间隔物”模型。通过生物物理研究，我们了解到半胱氨酸在氧化条件下促进液-液相分离，并通过二硫键交联使液体凝聚物持久存在，而二硫键交联可以通过氧化还原化学进行可逆调节。通过改变半胱氨酸的组成，观察到凝聚物的粘弹性行为有细微但明显的变化。根据经验，我们得出结论，半胱氨酸既不是“贴纸”也不是“间隔物”，而是作为共价节点来降低“贴纸”相互作用的有效浓度并抑制跨系统的渗流网络。我们共同揭示了半胱氨酸在蛋白质相行为中的作用以及开发可调节的、对氧化还原敏感的粘弹性材料的潜力。
