Understanding the relationship between a polypeptide sequence and its phase separation has important implications for analysing cellular function, treating disease and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid–liquid phase separation (LLPS), schematized as a ‘molecular grammar’ for LLPS. Here we further probe how sequence modulates phase separation and the material properties of the resulting condensates, targeting sequence features previously overlooked in the literature. We generate sequence variants of a repeat polypeptide with either no charged residues, high net charge, no glycine residues or devoid of aromatic or arginine residues. All but one of 12 variants exhibited LLPS, albeit to different extents, despite substantial differences in composition. Furthermore, we find that all the condensates formed behaved like viscous fluids, despite large differences in their viscosities. Our results support the model of multiple interactions between diverse residue pairs—not just a handful of residues—working in tandem to drive the phase separation and dynamics of condensates.

了解多肽序列与其相分离之间的关系对于分析细胞功能、治疗疾病和设计新型生物材料具有重要意义。一些序列特征已被确定为蛋白质液-液相分离 (LLPS) 的驱动因素，被图示为 LLPS 的“分子语法”。在这里，我们进一步探讨序列如何调节相分离和所得冷凝物的材料特性，针对文献中先前忽视的序列特征。我们生成重复多肽的序列变体，其不带电荷残基、高净电荷、无甘氨酸残基或缺乏芳香族或精氨酸残基。除了 12 个变体中的一个之外，所有变体都表现出 LLPS，尽管程度不同，尽管成分存在显着差异。此外，我们发现所有形成的冷凝物都表现得像粘性流体，尽管它们的粘度差异很大。我们的结果支持不同残基对（而不仅仅是少数残基）之间的多重相互作用模型，它们协同作用以驱动凝聚物的相分离和动力学。