No abstract

The formation of biomolecular condensates through phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used broadly by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but possess an internal structure consisting of distinct sub-compartments with different compositions. Notably, condensates can contain compartments that are depleted in the biopolymers that make up the condensate. Here, we show that such double-emulsion condensates emerge via dynamically arrested phase transitions. The combination of a change in composition coupled with a slow response to this change can lead to the nucleation of biopolymer-poor droplets within the polymer-rich condensate phase. Our findings demonstrate that condensates with a complex internal architecture can arise from kinetic, rather than purely thermodynamic driving forces, and provide more generally an avenue to understand and control the internal structure of condensates in vitro and in vivo.

Biomolecular condensates form via spontaneous and driven phase transitions of multivalent proteins and nucleic acids. These macromolecules can be organized in spatially inhomogeneous ways that lead to multiple coexisting dense phases with distinct macromolecular interfaces. While considerable attention has focused on the physical driving forces that give rise to phase separation from bulk solutions, the interactions that underlie adsorption driven wetting transitions remain unclear. Here, we report that pyrimidine-rich RNAs function as adsorbents that enable cascades of wetting transitions that include partial and complete wetting of condensates formed by purine-rich RNAs. Computations show that macromolecules that are scaffolds of condensates are oriented perpendicular to condensate interfaces whereas adsorbents are oriented parallel to interfaces. Our results yield heuristics for the design of synthetic materials that can be based on RNA-rich condensates featuring bespoke interfaces and distinct local microenvironments created by the interplay between scaffolds versus adsorbents.

Biomolecular phase separation has emerged as a critical process for regulating cellular organisation and function. Modulators, ranging from ionic species to small molecule compounds and macromolecular binders, are key in controlling phase behaviour. The mechanisms of action of such modulators, however, have remained challenging to resolve as means to study protein-modulator interactions within condensates are lacking. Here we unravel biophysical mechanisms of protein phase separation modulation by applying a tie-line-gradient-based analysis approach to characterize the effects of modulators on the partitioning and interactions of species across the dense and dilute phases. Using the protein fused in sarcoma (FUS), we first characterize the impact of ionic species on FUS phase separation and find that potassium chloride (KCl) ions are preferentially excluded from condensates at low ionic strengths as they counteract intermolecular interactions by charge screening. Conversely, in the high salt reentrant phase separation regime, salts preferentially partition into the dense phase thereby scaffolding condensates by enhancing non-ionic interactions. Furthermore, we show that the common phase separation disruptor 1,6-hexanediol decreases FUS phase separation propensity by acting as a solvation agent for the protein. This leads to an expansion of the polypeptide chain, which limits the ability of FUS to form inter- and intramolecular interactions, thereby counteracting condensation. Lastly, we study a sequence-specific modulator of FUS phase separation using a FUS-targeting RNA aptamer and find a concentration-dependent response of condensate stabilisation already at nanomolar concentrations. The sequence-specific binder first partitions as a client molecule into the condensate and then acts as a scaffolder within the condensate. Taken together, our study highlights a broadly applicable approach for studying condensate partitioning allowing us to decipher the mechanisms of action of phase separation modulators.

Solubility is a property of central importance for the use of proteins in research in molecular and cell biology and in applications in biotechnology and medicine. Since experimental methods for measuring protein solubility are material intensive and time consuming, computational methods have recently emerged to enable the rapid and inexpensive screening of solubility for large libraries of proteins, as it is routinely required in development pipelines. Here, we describe the development of one such method to include in the predictions the effect of the pH on solubility. We illustrate the resulting pH-dependent predictions on a variety of antibodies and other proteins to demonstrate that these predictions achieve an accuracy comparable with that of experimental methods. We make this method publicly available at https://www-cohsoftware.ch.cam.ac.uk/index.php/camsolph, as the version 3.0 of CamSol.