One of the key mechanisms used by cells to control the spatiotemporal organization of their many components is the formation and dissolution of biomolecular condensates through liquid–liquid phase separation (LLPS). Using a minimal coarse-grained model that allows us to simulate thousands of interacting multivalent proteins, we investigate the physical parameters dictating the stability and composition of multicomponent biomolecular condensates. We demonstrate that the molecular connectivity of the condensed-liquid network—i.e., the number of weak attractive protein–protein interactions per unit of volume—determines the stability (e.g., in temperature, pH, salt concentration) of multicomponent condensates, where stability is positively correlated with connectivity. While the connectivity of scaffolds (biomolecules essential for LLPS) dominates the phase landscape, introduction of clients (species recruited via scaffold–client interactions) fine-tunes it by transforming the scaffold–scaffold bond network. Whereas low-valency clients that compete for scaffold–scaffold binding sites decrease connectivity and stability, those that bind to alternate scaffold sites not required for LLPS or that have higher-than-scaffold valencies form additional scaffold–client–scaffold bridges increasing stability. Proteins that establish more connections (via increased valencies, promiscuous binding, and topologies that enable multivalent interactions) support the stability of and are enriched within multicomponent condensates. Importantly, proteins that increase the connectivity of multicomponent condensates have higher critical points as pure systems or, if pure LLPS is unfeasible, as binary scaffold–client mixtures. Hence, critical points of accessible systems (i.e., with just a few components) might serve as a unified thermodynamic parameter to predict the composition of multicomponent condensates.

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Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multicomponent mixtures of biomolecules (e.g., proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g., in intermolecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g., temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli or emerge progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the progressive intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of aging. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically aging condensates with nonconservative intermolecular forces. Our nonequilibrium simulations of condensate aging predict that single-component condensates that are initially homogeneous and liquid like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age due to gradual and irreversible enhancement of interprotein interactions. The type of multiphase architecture is determined by the aging mechanism, the molecular organization of the gel and liquid phases, and the chemical makeup of the protein. Notably, we predict that interprotein disorder to order transitions within the prion-like domains of intracellular proteins can lead to the required nonconservative enhancement of intermolecular interactions. Our study, therefore, predicts a potential mechanism by which the nonequilibrium process of aging results in single-component multiphase condensates.

Liquid–liquid phase separation (LLPS) is an important mechanism that helps explain the membraneless compartmentalization of the nucleus. Because chromatin compaction and LLPS are collective phenomena, linking their modulation to the physicochemical features of nucleosomes is challenging. Here, we develop an advanced multiscale chromatin model—integrating atomistic representations, a chemically-specific coarse-grained model, and a minimal model—to resolve individual nucleosomes within sub-Mb chromatin domains and phase-separated systems. To overcome the difficulty of sampling chromatin at high resolution, we devise a transferable enhanced-sampling Debye-length replica-exchange molecular dynamics approach. We find that nucleosome thermal fluctuations become significant at physiological salt concentrations and destabilize the 30-nm fiber. Our simulations show that nucleosome breathing favors stochastic folding of chromatin and promotes LLPS by simultaneously boosting the transient nature and heterogeneity of nucleosome–nucleosome contacts, and the effective nucleosome valency. Our work puts forward the intrinsic plasticity of nucleosomes as a key element in the liquid-like behavior of nucleosomes within chromatin, and the regulation of chromatin LLPS.

Intracellular liquid-liquid phase separation (LLPS) enables the formation of biomolecular condensates, which play a crucial role in the spatiotemporal organisation of biomolecules (proteins, oligonucleotides). While LLPS of biopolymers has been demonstrated in both experiments and computer simulations, the physical determinants governing phase separation of protein-oligonucleotide systems are not fully understood. Here, we introduce a minimal coarse-grained model to investigate concentration-dependent features of protein-oligonucleotide mixtures. We demonstrate that adding oligonucleotides 1