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The synergic effect of water and biomolecules in intracellular phase separation


Phase separation has long been observed within aqueous mixtures of two or more different compounds, such as proteins, salts, polysaccharides and synthetic polymers. A growing body of experimental evidence indicates that phase separation also takes place inside living cells, where intrinsically disordered proteins and other molecules such as RNA are thought to assemble into membraneless organelles. These structures represent a new paradigm of intracellular organization and compartmentalization, in which biochemical processes can be coordinated in space and time. Two thermodynamic driving forces have been proposed for phase separation: the strengths of macromolecule–macromolecule and macromolecule–H2O interactions, and the perturbation of H2O structure about different macromolecules. In this Perspective, we propose that both driving forces act in a concerted manner to promote phase separation, which we describe in the context of the well-known structural dynamics of intrinsically disordered proteins in the cellular milieu. We further suggest that this effect can be extended to explain how the partial unfolding of globular proteins can lead to intracellular phase separation.

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Fig. 1: Solutions can undergo liquid–liquid phase separation to afford aqueous two-phase systems.
Fig. 2: Hydration and bulk H2O around a solute.
Fig. 3: There are different types of aqueous polymer phase separation in biology.
Fig. 4: Free energy diagram for liquid–liquid phase separation and protein aggregation.
Fig. 5: Phase separation inside cells is driven by intermolecular interactions and H2O entropy.
Fig. 6: Thermally driven phase separation of partially unfolded globular proteins.


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The authors acknowledge Michael Smith for language advice. S.S.R., N.S. and S.E. acknowledge funding from the Human Frontier Science Program (HFSP; RGP0022/2017), Deutsche Forschungsgemeinschaft (German Research Foundation; SPP 2191) and the German–Israeli Foundation for Scientific Research and Development (grant 1410). J.C.M. acknowledges the Foundation for Science and Technology (FCT), Portugal, for financial support through the Centre of Chemistry of the University of Minho (CQ-UM) (project UID/QUI/00686/2016).

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Supplementary information


Amyloid fibrils

Highly ordered structures that result from protein aggregation and oligomer formation or association. These structures are bound together by interactions between β sheets. They are associated with several neurodegenerative diseases, such as Parkinson disease, amyotrophic lateral sclerosis and Alzheimer disease.


A liquid–liquid, phase-separation process that leads to the formation of a colloidal phase of concentrated solutions of charged or neutral molecules, including synthetic polymers, polyelectrolytes, polysaccharides and proteins.


Polymers and proteins that can be used in vitro to mimic the highly concentrated and heterogeneous environment within cells.


When facing conditions that are not ideal for growing, cells arrest their division cycle, entering a dormant state that involves biomolecular reorganization and diminished metabolic activity.


Solutes composed by both hydrophobic and hydrophilic sequences that solubilize hydrophobic compounds in water.

Intrinsically disordered protein

(IDP). A protein that does not have a well-defined 3D structure and exhibits high structural flexibility.

Intrinsically disordered protein region

(IDPR). A region within a protein that does not have a well-defined 3D structure and exhibits high structural flexibility.

Liquid–liquid phase separation

(LLPS). A process that involves two solutes demixing and forming two new phases of different composition. This is thought to be the basis for the formation of membraneless organelles.

Liquid-to-solid phase transition

(LSPT). Under ageing or stress conditions, the liquid compartments of membraneless organelles can change to a different, solid phase because their components (proteins) aggregate and eventually form amyloids.

Membraneless organelle

(MLO). An intracellular compartment without a membrane, formed through phase separation due to the heterogeneous distribution of biomolecules. It exhibits a liquid nature and provides a microenvironment that can serve a defined function, such as RNA metabolism.


A small molecule, such as a polyol, amino acid or methylamine, that alters protein folding and stability under osmotic stress conditions.

Protein aggregation

A phenomenon involving intermolecular interactions between misfolded proteins. This is usually the origin of amyloid formation and consequent diseases.

Quinary interactions

Weak, specific and transient interactions between proteins and other biomolecules that appear to have a crucial function in cellular organization.

Solvent regime

A polymer chemistry concept that reflects the favourability of polymer chains with the solvent relative to chain–solvent and solvent–solvent interactions. In the poor solvent regime, intramolecular and intermolecular interactions of the polymer are favoured in comparison with chain–solvent interactions and, so, the solvent is considered poor. The inverse situation applies in the good solvent regime.

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Ribeiro, S.S., Samanta, N., Ebbinghaus, S. et al. The synergic effect of water and biomolecules in intracellular phase separation. Nat Rev Chem 3, 552–561 (2019).

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