Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, United States.
Department of Physics, University of Illinois at Chicago, Chicago, Illinois 60607, United States.
J Am Chem Soc. 2020 May 13;142(19):8848-8861. doi: 10.1021/jacs.0c01881. Epub 2020 May 4.
Membraneless organelles, comprising dozens to hundreds of macromolecular components, form heterogeneous phases in space and evolve over time in material properties. Here, using four macromolecules, we demonstrate a range of phase behaviors associated with membraneless organelles and uncover the underlying physicochemical rules. The macromolecules are SH3 (S) and PRM (P), two pentameric, oppositely charged protein constructs; heparin (H), an anionic polymer; and lysozyme (L), a cationic single-domain protein. The S:P, S:L, and P:H binaries form droplets, but the H:L binary forms network-like precipitates, therefore setting up a tug of war between different condensate phases within the S:P:H:L quaternary. The H:L exception can partly be attributed to the compactness of L, as supported by ThT binding data. Increasing amounts of P alone or both S and P, but not S alone, can dissolve H:L precipitates into droplets. These differential effects can be explained by the order of the strengths of pairwise attraction: H:L > P:H > S:P > S:L, deduced from the shapes of ternary phase boundaries. When S and P are at subdissolution concentrations, S:P:H:L precipitates change over time to become droplet-like in appearance, although not completely fluidic according to fluorescence recovery after photobleaching. In fact, confocal microscopy reveals separated S:L-rich and P:H-rich foci inside the droplet-like condensates. Therefore, complex phase behaviors of membraneless organelles, including rescue of aberrant phase transitions, demixing of condensates, and time evolution of material properties, can all be reconstituted and understood via a minimal macromolecular system.
无膜细胞器由数十到数百种大分子成分组成，在空间中形成异质相，并在物质性质上随时间演变。在这里，我们使用四种大分子展示了一系列与无膜细胞器相关的相行为，并揭示了潜在的物理化学规律。这四种大分子是 SH3（S）和 PRM（P），两种五聚体、带相反电荷的蛋白质构建体；肝素（H），一种阴离子聚合物；和溶菌酶（L），一种阳离子单域蛋白。S:P、S:L 和 P:H 二元混合物形成液滴，但 H:L 二元混合物形成网状沉淀物，因此在 S:P:H:L 四元混合物内引发了不同凝聚相之间的拔河比赛。H:L 异常部分可以归因于 L 的紧凑性，这得到 ThT 结合数据的支持。单独增加 P 的量或同时增加 S 和 P，但不是 S，都可以将 H:L 沉淀物溶解成液滴。这些不同的效应可以通过成对吸引力强度的顺序来解释：H:L > P:H > S:P > S:L，这是从三元相界的形状推断出来的。当 S 和 P 处于亚溶解浓度时，S:P:H:L 沉淀物随时间变化而变为类似液滴的外观，尽管根据光漂白后荧光恢复，它们不完全是流体。事实上，共聚焦显微镜揭示了液滴状凝聚物内分离的 S:L 丰富和 P:H 丰富的焦点。因此，无膜细胞器的复杂相行为，包括异常相转变的挽救、凝聚物的分相以及物质性质的时间演变，都可以通过最小的大分子系统来重建和理解。
