Multiphase Organization Is a Second Phase Transition Within Multi-Component Biomolecular Condensates

Biomolecular condensates formed via phase separation are a very common occurrence in vivo and in vitro. When multiple components are present, condensates often demix to form multiple phases. Using a mean-field theoretical model and molecular simulations, we show that the multiphase organization is a second phase transition. Whereas the first phase transition that results in the separation of condensates from the bulk phase is driven by overall attraction among the macromolecular components, the second phase transition, leading to multiphase organization within condensates, is driven by disparity in strength between self and cross-species attraction. At a given strength of cross-species attraction, both of the phase transitions can be observed by decreasing temperature, leading first to phase separation and then to demixing of the condensates. These predictions are validated by molecular dynamics simulations of model binary mixtures. The binary mixtures comprise two types of Lennard-Jones particles or chains: D and R, with strengths of self-attraction ε_DD and ε_RR. At temperatures below the critical temperature for phase separation and strengths of cross-species attraction (ε_DR) above the mean of ε_DD and ε_RR, the components are homogeneously mixed in the condensates. When ε_DR is below the mean of ε_DD and ε_RR, condensates demix when temperatures are below a second critical value. The demixing leads to a variety of multiphase configurations, including core-shell, physical association, and droplet-inside-droplet. Calculation of interfacial tensions suggest that some of these configurations are metastable. Together, these results provide the missing physical underpinning for the multiphase organization of biomolecular condensates.