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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Tolstoguzov, V. Origins of globular structure in proteins. FEBS Lett. 444, 145–148 (1999).
Polyakov, V. I., Grinberg, Ya. V. & Tolstoguzov, V. B. Thermodynamic incompatibility of proteins. Food Hydrocoll. 11, 171–180 (1997).
Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).
Franzmann, T. M. & Alberti, S. Protein phase separation as a stress survival strategy. Cold Spring Harb. Perspect. Biol. 11, a034058 (2019).
Li, X.-H. et al. Function and regulation of phase-separated biological condensates. Biochemistry 57, 2452–2461 (2018).
van der Lee, R. et al. Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589–6631 (2014).
Nott, T. J., Craggs, T. D. & Baldwin, A. J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 8, 569–575 (2016).
Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
Brangwynne, C. P., Mitchison, T. J. & Hyman, A. A. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc. Natl Acad. Sci. USA 108, 4334–4339 (2011).
Saito, M. et al. Acetylation of intrinsically disordered regions regulates phase separation. Nat. Chem. Biol. 15, 51–61 (2019).
Ferreon, J. C. et al. Acetylation disfavors tau phase separation. Int. J. Mol. Sci. 19, 1360 (2018).
Monahan, Z. et al. Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J. 36, 2951–2967 (2017).
Guillén-Boixet, J. et al. CPEB4 is regulated during cell cycle by ERK2/Cdk1-mediated phosphorylation and its assembly into liquid-like droplets. eLife 5, e19298 (2016).
Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015).
Riback, J. A. et al. Stress-triggered phase separation is an adaptive, evolutionarily tuned response. Cell 168, 1028–1040 (2017).
Kroschwald, S. et al. Different material states of Pub1 condensates define distinct modes of stress adaptation and recovery. Cell Rep. 23, 3327–3339 (2018).
Franzmann, T. M. et al. Phase separation of a yeast prion protein promotes cellular fitness. Science 359, eaao5654 (2018).
Wirth, A. J. & Gruebele, M. Quinary protein structure and the consequences of crowding in living cells: leaving the test-tube behind. Bioessays 35, 984–993 (2013).
Chien, P. & Gierasch, L. M. Challenges and dreams: physics of weak interactions essential to life. Mol. Biol. Cell 25, 3474–3477 (2014).
Ribeiro, S., Ebbinghaus, S. & Marcos, J. C. Protein folding and quinary interactions: creating cellular organisation through functional disorder. FEBS Lett. 592, 3040–3053 (2018).
Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).
Strom, A. R. et al. Phase separation drives heterochromatin domain formation. Nature 547, 241–245 (2017).
Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25, 833–840 (2018).
Lu, H. et al. Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558, 318–323 (2018).
Sheu-Gruttadauria, J. & MacRae, I. J. Phase transitions in the assembly and function of human miRISC. Cell 173, 946–957 (2018).
Fox, A. H. et al. Paraspeckles: where long noncoding RNA meets phase separation. Trends Biochem. Sci. 43, 124–135 (2018).
Trinkle-Mulcahy, L. & Sleeman, J. E. The Cajal body and the nucleolus: “in a relationship” or “it’s complicated”? RNA Biol. 14, 739–751 (2017).
Perez-Pepe, M., Fernández-Alvarez, A. J. & Boccaccio, G. L. Life and work of stress granules and processing bodies: new insights into their formation and function. Biochemistry 57, 2488–2498 (2018).
Antonicka, H. & Shoubridge, E. A. Mitochondrial RNA granules are centers for posttranscriptional RNA processing and ribosome biogenesis. Cell Rep. 10, 920–932 (2015).
Uniacke, J. & Zerges, W. Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii. J. Cell Biol. 182, 641–646 (2008).
Alberti, S. & Carra, S. Quality control of membraneless organelles. J. Mol. Biol. 430, 4711–4729 (2018).
Mateju, D. et al. An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function. EMBO J. 36, 1669–1687 (2017).
Naumann, M. et al. Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation. Nat. Commun. 9, 335 (2018).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
Beijerinck, M. W. Über eine Eigentümlichkeit der löslichen Stärke. Zentralbl. Bakteriol. 2, 697–699 (1896).
Albertsson, P.-Å. Chromatography and partition of cells and cell fragments. Nature 177, 771–774 (1956).
Albertsson, P.-Å. Partition of proteins in liquid polymer–polymer two-phase systems. Nature 182, 709–711 (1958).
Lee, S. Y. et al. Recent advances in protein extraction using ionic liquid-based aqueous two-phase systems. Sep. Purif. Rev. 46, 291–304 (2017).
Tolstoguzov, V. Compositions and phase diagrams for aqueous systems based on proteins and polysaccharides. Int. Rev. Cytol. 192, 3–31 (1999).
Albertsson, P.-Å. Partition of Cell Particles and Macromolecules 3rd edn (Wiley-Blackwell, Weinheim, 1986).
Torres-Acosta, M. A. et al. Aqueous two-phase systems at large scale: challenges and opportunities. Biotechnol. J. 14, 1800117 (2019).
González-González, M. & Ruiz-Ruiz, F. in Aqueous Two-Phase Systems for Bioprocess Development for the Recovery of Biological Products (eds Rito-Palomares, M. & Benavides, J.) 55–78 (Springer, Cham, 2017).
Teixeira, A. G. et al. Emerging biotechnology applications of aqueous two-phase systems. Adv. Healthcare Mater. 7, 1701036 (2018).
Helfrich, M. R. et al. Aqueous phase separation in giant vesicles. J. Am. Chem. Soc. 124, 13374–13375 (2002).
Long, M. S. et al. Dynamic microcompartmentation in synthetic cells. Proc. Natl Acad. Sci. USA 102, 5920–5925 (2005).
Long, M. S., Cans, A.-S. & Keating, C. D. Budding and asymmetric protein microcompartmentation in giant vesicles containing two aqueous phases. J. Am. Chem. Soc. 130, 756–762 (2008).
Zaslavsky, B. Y. Aqueous Two-phase Partitioning: Physical Chemistry and Bioanalytical Applications (Marcel Dekker, New York, 1995).
Walter, H. & Brooks, D. E. Phase separation in cytoplasm, due to macromolecular crowding, is the basis for microcompartmentation. FEBS Lett. 361, 135–139 (1995).
Uversky, V. N. Protein intrinsic disorder-based liquid–liquid phase transitions in biological systems: complex coacervates and membrane-less organelles. Adv. Colloid Interface Sci. 239, 97–114 (2017).
Flory, P. J. Thermodynamics of high polymer solutions. J. Chem. Phys. 10, 51–61 (1942).
Huggins, M. L. Thermodynamic properties of solutions of long-chain compounds. Ann. NY Acad. Sci. 43, 1–32 (1942).
Flory, P. J. Principles of Polymer Chemistry 672 (Cornell Univ. Press, Ithaca, 1953).
Zaslavsky, B. Y. et al. Structure of water as a key factor of phase separation in aqueous mixtures of two nonionic polymers. Polymer 30, 2104–2111 (1989).
Zaslavsky, B. Y. et al. Aqueous biphasic systems formed by nonionic polymers I. Effects of inorganic salts on phase separation. Colloid Polym. Sci. 264, 1066–1071 (1986).
Zaslavsky, B. Y. et al. The solvent side of proteinaceous membrane-less organelles in light of aqueous two-phase systems. Int. J. Biol. Macromol. 117, 1224–1251 (2018).
Zaslavsky, B. Y. & Uversky, V. N. In aqua veritas: the indispensable yet mostly ignored role of water in phase separation and membrane-less organelles. Biochemistry 57, 2437–2451 (2018).
Ball, P. Water is an active matrix of life for cell and molecular biology. Proc. Natl Acad. Sci. USA 114, 13327–13335 (2017).
Das Mahanta, D. et al. Non-monotonic dynamics of water in its binary mixture with 1,2-dimethoxy ethane: a combined THz spectroscopic and MD simulation study. J. Chem. Phys. 145, 164501 (2016).
Samanta, N., Das Mahanta, D. & Kumar Mitra, R. Does urea alter the collective hydrogen-bond dynamics in water? A dielectric relaxation study in the terahertz-frequency region. Chem. Asian J. 9, 3457–3463 (2014).
Eaves, J. D. et al. Hydrogen bonds in liquid water are broken only fleetingly. Proc. Natl Acad. Sci. USA 102, 13019–13022 (2005).
Perticaroli, S. et al. Description of hydration water in protein (green fluorescent protein) solution. J. Am. Chem. Soc. 139, 1098–1105 (2017).
Tan, P. et al. Gradual crossover from subdiffusion to normal diffusion: a many-body effect in protein surface water. Phys. Rev. Lett. 120, 248101 (2018).
Aumiller, W. M. & Keating, C. D. Experimental models for dynamic compartmentalization of biomolecules in liquid organelles: reversible formation and partitioning in aqueous biphasic systems. Adv. Colloid Interface Sci. 239, 75–87 (2017).
Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016).
Veis, A. A review of the early development of the thermodynamics of the complex coacervation phase separation. Adv. Colloid Interface Sci. 167, 2–11 (2011).
Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell 63, 72–85 (2016).
Burke, K. A. et al. Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase ii. Mol. Cell 60, 231–241 (2015).
Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl Acad. Sci. USA 112, 7189–7194 (2015).
Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015).
Banani, S. F. et al. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell. Biol. 18, 285–298 (2017).
Simon, J. R. et al. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity. Nat. Chem. 9, 509–515 (2017).
Uversky, V. N. Intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder. Curr. Opin. Struct. Biol. 44, 18–30 (2017).
Tombs, M. P., Newsom, B. G. & Wilding, P. Protein solubility: phase separation in arachin-salt-water systems. Int. J. Pept. Protein Res. 6, 253–277 (1974).
Tanaka, T., Ishimoto, C. & Chylack, L. T. Phase separation of a protein–water mixture in cold cataract in the young rat lens. Science 197, 1010–1012 (1977).
Tanaka, S., Ataka, M. & Ito, K. Pattern formation and coarsening during metastable phase separation in lysozyme solutions. Phys. Rev. E 65, 051804 (2002).
Dumetz, A. C. et al. Protein phase behavior in aqueous solutions: crystallization, liquid-liquid phase separation, gels, and aggregates. Biophys. J. 94, 570–583 (2008).
McManus, J. J. et al. Altered phase diagram due to a single point mutation in human γD-crystallin. Proc. Natl Acad. Sci. USA 104, 16856–16861 (2007).
Pande, A. et al. Molecular basis of a progressive juvenile-onset hereditary cataract. Proc. Natl Acad. Sci. USA 97, 1993–1998 (2000).
Bergeron-Sandoval, L.-P., Safaee, N. & Michnick, S. W. Mechanisms and consequences of macromolecular phase separation. Cell 165, 1067–1079 (2016).
Zhou, H.-X. et al. Why do disordered and structured proteins behave differently in phase separation? Trends Biochem. Sci. 43, 499–516 (2018).
Kaganovich, D. There is an inclusion for that: material properties of protein granules provide a platform for building diverse cellular functions. Trends Biochem. Sci. 42, 765–776 (2017).
Owen, M. C. et al. Effects of in vivo conditions on amyloid aggregation. Chem. Soc. Rev. https://doi.org/10.1039/c8cs00034d (2019).
Scott, R. L. The thermodynamics of high polymer solutions. V. Phase equilibria in the ternary system: polymer 1–polymer 2–solvent. J. Chem. Phys. 17, 279–284 (1949).
Gustafsson, Å., Wennerström, H. & Tjerneld, F. The nature of phase separation in aqueous two-polymer systems. Polymer 27, 1768–1770 (1986).
Brady, J. P. et al. Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. Proc. Natl Acad. Sci. USA 114, E8194–E8203 (2017).
Lee, C. F. et al. Spatial organization of the cell cytoplasm by position-dependent phase separation. Phys. Rev. Lett. 111, 088101 (2013).
Saha, S. et al. Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism. Cell 166, 1572–1584 (2016).
Lin, Y.-H., Forman-Kay, J. D. & Chan, H. S. Theories for sequence-dependent phase behaviors of biomolecular condensates. Biochemistry 57, 2499–2508 (2018).
Overbeek, J. T. G. & Voorn, M. J. Phase separation in polyelectrolyte solutions. Theory of complex coacervation. J. Cell. Comp. Physiol. 49, 7–26 (1957).
Lin, Y.-H. et al. Charge pattern matching as a ‘fuzzy’ mode of molecular recognition for the functional phase separations of intrinsically disordered proteins. New J. Phys. 19, 115003 (2017).
Ferreira, A. L., Uversky, N. V. & Zaslavsky, Y. B. Role of solvent properties of water in crowding effects induced by macromolecular agents and osmolytes. Mol. Biosyst. 13, 2551–2563 (2017).
Ferreira, L. A. et al. Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn. 34, 92–103 (2016).
Ferreira, L. A., Uversky, V. N. & Zaslavsky, B. Y. Effects of the Hofmeister series of sodium salts on the solvent properties of water. Phys. Chem. Chem. Phys. 19, 5254–5261 (2017).
Ferreira, L. A., Uversky, V. N. & Zaslavsky, B. Y. Modified binodal model describes phase separation in aqueous two-phase systems in terms of the effects of phase-forming components on the solvent features of water. J. Chromatogr. A 1567, 226–232 (2018).
da Silva, N. R. et al. Effects of sodium chloride and sodium perchlorate on properties and partition behavior of solutes in aqueous dextran-polyethylene glycol and polyethylene glycol–sodium sulfate two-phase systems. J. Chromatogr. A 1583, 28–38 (2019).
Ferreira, L. A. et al. Effect of human heat shock protein HspB6 on the solvent features of water in aqueous solutions. J. Biomol. Struct. Dyn. 36, 1520–1528 (2018).
Ferreira, L. A. et al. Effect of an intrinsically disordered plant stress protein on the properties of water. Biophys. J. 115, 1696–1706 (2018).
Ferreira, L. A., Uversky, V. N. & Zaslavsky, B. Y. Effects of amino acids on solvent properties of water. J. Mol. Liq. 277, 123–131 (2019).
Zimmerman, S. B. & Trach, S. O. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 222, 599–620 (1991).
Bellissent-Funel, M.-C. et al. Water determines the structure and dynamics of proteins. Chem. Rev. 116, 7673–7697 (2016).
Ebbinghaus, S. et al. An extended dynamical hydration shell around proteins. Proc. Natl Acad. Sci. USA 104, 20749–20752 (2007).
Born, B. et al. The terahertz dance of water with the proteins: the effect of protein flexibility on the dynamical hydration shell of ubiquitin. Faraday Discuss. 141, 161–173 (2008).
Persson, F., Söderhjelm, P. & Halle, B. The spatial range of protein hydration. J. Chem. Phys. 148, 215104 (2018).
Harada, R., Sugita, Y. & Feig, M. Protein crowding affects hydration structure and dynamics. J. Am. Chem. Soc. 134, 4842–4849 (2012).
Persson, E. & Halle, B. Cell water dynamics on multiple time scales. Proc. Natl Acad. Sci. USA 105, 6266–6271 (2008).
Tros, M. et al. Picosecond orientational dynamics of water in living cells. Nat. Commun. 8, 904 (2017).
Vekilov, P. G. Phase transitions of folded proteins. Soft Matter 6, 5254–5272 (2010).
Pal, S. K. & Zewail, A. H. Dynamics of water in biological recognition. Chem. Rev. 104, 2099–2124 (2004).
Rani, P. & Biswas, P. Diffusion of hydration water around intrinsically disordered proteins. J. Phys. Chem. B 119, 13262–13270 (2015).
Rani, P. & Biswas, P. Local structure and dynamics of hydration water in intrinsically disordered proteins. J. Phys. Chem. B 119, 10858–10867 (2015).
Arya, S. & Mukhopadhyay, S. Ordered water within the collapsed globules of an amyloidogenic intrinsically disordered protein. J. Phys. Chem. B 118, 9191–9198 (2014).
Arya, S. et al. Water rearrangements upon disorder-to-order amyloid transition. J. Phys. Chem. Lett. 7, 4105–4110 (2016).
Arya, S. et al. Femtosecond hydration map of intrinsically disordered α-synuclein. Biophys. J. 114, 2540–2551 (2018).
Thirumalai, D., Reddy, G. & Straub, J. E. Role of water in protein aggregation and amyloid polymorphism. Acc. Chem. Res. 45, 83–92 (2012).
Knowles, T. P. J., Vendruscolo, M. & Dobson, C. M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell. Biol. 15, 384–396 (2014).
Chatani, E. & Yamamoto, N. Recent progress on understanding the mechanisms of amyloid nucleation. Biophys. Rev. 10, 527–534 (2018).
Reddy, G., Straub, J. E. & Thirumalai, D. Dynamics of locking of peptides onto growing amyloid fibrils. Proc. Natl Acad. Sci. USA 106, 11948–11953 (2009).
Reddy, G., Straub, J. E. & Thirumalai, D. Dry amyloid fibril assembly in a yeast prion peptide is mediated by long-lived structures containing water wires. Proc. Natl Acad. Sci. USA 107, 21459–21464 (2010).
Fichou, Y. et al. Hydration water mobility is enhanced around tau amyloid fibers. Proc. Natl Acad. Sci. USA 112, 6365–6370 (2015).
Wegmann, S. et al. Tau protein liquid–liquid phase separation can initiate tau aggregation. EMBO J. 37, e98049 (2018).
Pavlova, A. et al. Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau. Proc. Natl Acad. Sci. USA 113, E127–E136 (2016).
Patel, A. et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).
Ambadipudi, S. et al. Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein tau. Nat. Commun. 8, 275 (2017).
Boyko, S. et al. Liquid–liquid phase separation of tau protein: the crucial role of electrostatic interactions. J. Biol. Chem. https://doi.org/10.1074/jbc.AC119.009198 (2019).
Ray, S. et al. Liquid–liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/619858v1 (2019).
Reichheld, S. E. et al. Direct observation of structure and dynamics during phase separation of an elastomeric protein. Proc. Natl Acad. Sci. USA 114, E4408–E4415 (2017).
Rauscher, S. & Pomès, R. The liquid structure of elastin. eLife 6, e26526 (2017).
Lin, Y. et al. Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions. eLife 8, e42571 (2019).
Patel, A. et al. ATP as a biological hydrotrope. Science 356, 753–756 (2017).
Jain, A. & Vale, R. D. RNA phase transitions in repeat expansion disorders. Nature 546, 243–247 (2017).
Langdon, E. M. et al. mRNA structure determines specificity of a polyQ-driven phase separation. Science 360, 922–927 (2018).
Van Treeck, B. et al. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome. Proc. Natl Acad. Sci. USA 115, 2734–2739 (2018).
Van Treeck, B. & Parker, R. Emerging roles for intermolecular RNA–RNA interactions in RNP assemblies. Cell 174, 791–802 (2018).
Yoon, J. et al. Dynamical transition and heterogeneous hydration dynamics in RNA. J. Phys. Chem. B 118, 7910–7919 (2014).
Lammert, H. et al. RNA as a complex polymer with coupled dynamics of ions and water in the outer solvation sphere. J. Phys. Chem. B 122, 11218–11227 (2018).
Denisov, V. P. et al. Orientational disorder and entropy of water in protein cavities. J. Phys. Chem. B 101, 9380–9389 (1997).
Chong, S.-H. & Ham, S. Dynamics of hydration water plays a key role in determining the binding thermodynamics of protein complexes. Sci. Rep. 7, 8744 (2017).
Broide, M. L., Tominc, T. M. & Saxowsky, M. D. Using phase transitions to investigate the effect of salts on protein interactions. Phys. Rev. E 53, 6325–6335 (1996).
Möller, J. et al. Reentrant liquid-liquid phase separation in protein solutions at elevated hydrostatic pressures. Phys. Rev. Lett. 112, 028101 (2014).
Barnett, G. V. et al. Osmolyte effects on monoclonal antibody stability and concentration-dependent protein interactions with water and common osmolytes. J. Phys. Chem. B 120, 3318–3330 (2016).
Annunziata, O. et al. Effect of polyethylene glycol on the liquid–liquid phase transition in aqueous protein solutions. Proc. Natl Acad. Sci. USA 99, 14165–14170 (2002).
Protter, D. S. W. et al. Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly. Cell Rep. 22, 1401–1412 (2018).
Kaur, T. et al. Molecular crowding tunes material states of ribonucleoprotein condensates. Biomolecules 9, 71 (2019).
Samanta, N. et al. Short chain polyethylene glycols unusually assist thermal unfolding of human serum albumin. Biochimie 104, 81–89 (2014).
Verma, P. K. et al. Role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin under crowded environment. Biochimie 93, 1424–1433 (2011).
Zhang, J. in Protein–Protein interactions — Computational and Experimental Tools (eds Cai, W. & Hong, H.) 359–376 (InTechOpen, 2012).
Urry, D. W. Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics. J. Protein Chem. 7, 1–34 (1988).
Li, N. K. et al. Molecular description of the LCST behavior of an elastin-like polypeptide. Biomacromolecules 15, 3522–3530 (2014).
Wuttke, R. et al. Temperature-dependent solvation modulates the dimensions of disordered proteins. Proc. Natl Acad. Sci. USA 111, 5213–5218 (2014).
Wallace, E. W. J. et al. Reversible, specific, active aggregates of endogenous proteins assemble upon heat stress. Cell 162, 1286–1298 (2015).
Narayanaswamy, R. et al. Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation. Proc. Natl Acad. Sci. USA 106, 10147–10152 (2009).
Petrovska, I. et al. Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation. eLife 3, e02409 (2014).
Munder, M. C. et al. A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy. eLife 5, e09347 (2016).
Kuffel, A. How water mediates the long-range interactions between remote protein molecules. Phys. Chem. Chem. Phys. 19, 5441–5448 (2017).
Samanta, N. et al. Effect of short chain poly(ethylene glycol)s on the hydration structure and dynamics around human serum albumin. Langmuir 32, 831–837 (2016).
King, J. T. et al. Crowding induced collective hydration of biological macromolecules over extended distances. J. Am. Chem. Soc. 136, 188–194 (2014).
Aggarwal, L. & Biswas, P. Hydration water distribution around intrinsically disordered proteins. J. Phys. Chem. B 122, 4206–4218 (2018).
Ditlev, J. A., Case, L. B. & Rosen, M. K. Who’s in and who’s out — compositional control of biomolecular condensates. J. Mol. Biol. 430, 4666–4684 (2018).
Wang, T. et al. Water distribution, dynamics, and interactions with Alzheimer’s β-amyloid fibrils investigated by solid-state NMR. J. Am. Chem. Soc. 139, 6242–6252 (2017).
Yang, X. et al. Biomedical applications of terahertz spectroscopy and imaging. Trends Biotechnol. 34, 810–824 (2016).
Shiraga, K. et al. Hydration state inside HeLa cell monolayer investigated with terahertz spectroscopy. Appl. Phys. Lett. 106, 253701 (2015).
Li, H.-R. et al. TAR DNA-binding protein 43 (TDP-43) liquid–liquid phase separation is mediated by just a few aromatic residues. J. Biol. Chem. 293, 6090–6098 (2018).
Vanderweyde, T. et al. Interaction of tau with the RNA-binding protein TIA1 regulates tau pathophysiology and toxicity. Cell Rep. 15, 1455–1466 (2016).
Luo, Y., Na, Z. & Slavoff, S. A. P-bodies: composition, properties, and functions. Biochemistry 57, 2424–2431 (2018).
Protter, D. S. W. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).
Nakagawa, S., Yamazaki, T. & Hirose, T. Molecular dissection of nuclear paraspeckles: towards understanding the emerging world of the RNP milieu. Open Biol. 8, 180150 (2018).
Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).
Li, H.-R. et al. The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43. Biochim. Biophys. Acta 1866, 214–223 (2018).
Ryan, V. H. et al. Mechanistic view of hnRNPA2 low-complexity domain structure, interactions, and phase separation altered by mutation and arginine methylation. Mol. Cell 69, 465–479 (2018).
Dao, T. P. et al. Ubiquitin modulates liquid–liquid phase separation of UBQLN2 via disruption of multivalent interactions. Mol. Cell 69, 965–978 (2018).
Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).
Banjade, S. & Rosen, M. K. Phase transitions of multivalent proteins can promote clustering of membrane receptors. eLife 3, e04123 (2014).
Mitrea, D. M. et al. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. eLife 5, e13571 (2016).
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699 (2018).
Banerjee, P. R. et al. Reentrant phase transition drives dynamic substructure formation in ribonucleoprotein droplets. Angew. Chem. Int. Ed. 56, 11354–11359 (2017).
Maharana, S. et al. RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science 360, 918–921 (2018).
Nguemaha, V. & Zhou, H.-X. Liquid–liquid phase separation of patchy particles illuminates diverse effects of regulatory components on protein droplet formation. Sci. Rep. 8, 6728 (2018).
Kucherenko, M. M. & Shcherbata, H. R. miRNA targeting and alternative splicing in the stress response - events hosted by membrane-less compartments. J. Cell Sci. 131, jcs202002 (2018).
Conicella, A. E. et al. ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain. Structure 24, 1537–1549 (2016).
Brangwynne, C. P., Tompa, P. & Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 11, 899–904 (2015).
Semenov, A. N. & Rubinstein, M. Thermoreversible gelation in solutions of associative polymers. 1. Statics. Macromolecules 31, 1373–1385 (1998).
Holehouse, A. S. & Pappu, R. V. Functional implications of intracellular phase transitions. Biochemistry 57, 2415–2423 (2018).
Harmon, T. S. et al. Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. eLife 6, e30294 (2017).
Hofweber, M. et al. Phase separation of FUS is suppressed by its nuclear import receptor and arginine methylation. Cell 173, 706–719 (2018).
Kiebler, M. A. & Bassell, G. J. Neuronal RNA granules: movers and makers. Neuron 51, 685–690 (2006).
Banjade, S. et al. Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck. Proc. Natl Acad. Sci. USA 112, E6426–E6435 (2015).
Ferrolino, M. C. et al. Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms. Nat. Commun. 9, 5064 (2018).
Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353, aac4354 (2016).
Gallo, A. et al. Structure of nucleophosmin DNA-binding domain and analysis of its complex with a G-quadruplex sequence from the c-MYC promoter. J. Biol. Chem. 287, 26539–26548 (2012).
Wheeler, K. E. et al. Dynamic docking of cytochrome b5 with myoglobin and alpha-hemoglobin: heme-neutralization “squares” and the binding of electron-transfer-reactive configurations. J. Am. Chem. Soc. 129, 3906–3917 (2007).
Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).
Fourmy, D. et al. Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science 274, 1367–1371 (1996).
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).
The authors declare no competing interests.
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- 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). https://doi.org/10.1038/s41570-019-0120-4
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