Widespread occurrence of the droplet state of proteins in the human proteome

A wide range of proteins have been reported to condensate into a dense liquid phase, forming a reversible droplet state. Failure in the control of the droplet state can lead to the formation of the more stable amyloid state, which is often disease-related. These observations prompt the question of how many proteins can undergo liquid–liquid phase separation. Here, in order to address this problem, we discuss the biophysical principles underlying the droplet state of proteins by analyzing current evidence for droplet-driver and droplet-client proteins. Based on the concept that the droplet state is stabilized by the large conformational entropy associated with nonspecific side-chain interactions, we develop the FuzDrop method to predict droplet-promoting regions and proteins, which can spontaneously phase separate. We use this approach to carry out a proteome-level study to rank proteins according to their propensity to form the droplet state, spontaneously or via partner interactions. Our results lead to the conclusion that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.

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Widespread occurrence of the droplet state of proteins in the human proteome

10.1101/2020.10.21.348532 ◽

2020 ◽

Cited By ~ 2

Author(s):

Maarten Hardenberg ◽

Attila Horvath ◽

Viktor Ambrus ◽

Monika Fuxreiter ◽

Michele Vendruscolo

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Current Evidence ◽

Cellular Environment ◽

Wide Range ◽

Proteome Level ◽

Client Proteins ◽

Separation Of Proteins ◽

Liquid Liquid Phase Separation

AbstractA wide range of proteins have been reported to condensate into a dense liquid phase, forming a reversible droplet state. Failure in the control of the droplet state can lead to the formation of the more stable amyloid state, which is often disease-related. These observations prompt the question of how many proteins can undergo liquid-liquid phase separation. Here, in order to address this problem, we discuss the biophysical principles underlying the droplet state of proteins by analyzing current evidence for droplet-driver and droplet-client proteins. Based on the concept that the droplet state is stabilized by the large conformational entropy associated with non-specific side-chain interactions, we develop the FuzDrop method to predict droplet-promoting regions and proteins, which can spontaneously phase separate. We use this approach to carry out a proteome-level study to rank proteins according to their propensity to form the droplet state, spontaneously or via partner interactions. Our results lead to the conclusion that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.SignificanceLiquid-liquid phase separation of proteins results in biomolecular condensates, which contribute to the organisation of cellular matter into membraneless organelles. It is still unclear, however, whether these condensates represent a common state of proteins. Here, based on biophysical principles driving phase separation, we report a proteome-wide ranking of proteins according to their propensity to condensate into a droplet state. We analyze two mechanisms for droplet formation - driver proteins can spontaneously phase separate, while client proteins require additional components. We conclude that the droplet state, as the native and amyloid states, is a fundamental state of proteins, with most proteins expected to be capable of undergoing liquid-liquid phase separation via either of these two mechanisms.

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Fuzzy protein theory for disordered proteins

Biochemical Society Transactions ◽

10.1042/bst20200239 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2557-2564

Author(s):

Monika Fuxreiter

Keyword(s):

Fuzzy Logic ◽

Phase Separation ◽

Liquid Phase ◽

Disordered Proteins ◽

Cellular Behavior ◽

Alternative Pathways ◽

Conformational Ensembles ◽

Cellular Environment ◽

Wide Range ◽

Liquid Liquid Phase Separation

Why proteins are fuzzy? Constant adaptation to the cellular environment requires a wide range of changes in protein structure and interactions. Conformational ensembles of disordered proteins in particular exhibit large shifts to activate or inhibit alternative pathways. Fuzziness is critical for liquid–liquid phase separation and conversion of biomolecular condensates into fibrils. Interpretation of these phenomena presents a challenge for the classical structure-function paradigm. Here I discuss a multi-valued formalism, based on fuzzy logic, which can be applied to describe complex cellular behavior of proteins.

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Liquid-liquid phase separation and viscosity within secondary organic aerosol generated from diesel fuel vapors

10.5194/acp-2019-367 ◽

2019 ◽

Author(s):

Mijung Song ◽

Adrian M. Maclean ◽

Yuanzhou Huang ◽

Natalie R. Smith ◽

Sandra L. Blair ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Diesel Fuel ◽

Diffusion Coefficients ◽

Secondary Organic Aerosol ◽

Organic Aerosol ◽

Liquid Phase Separation ◽

Small Diffusion ◽

Wide Range ◽

Liquid Liquid Phase Separation

Abstract. Information on liquid-liquid phase separation (LLPS) and viscosity (or diffusion) within secondary organic aerosol (SOA) is needed to improve predictions of particle size, mass, reactivity, and cloud nucleating properties in the atmosphere. Here we report on LLPS and viscosities within SOA generated by the photooxidation of diesel fuel vapors. Diesel fuel contains a wide range of volatile organic compounds, and SOA generated by the photooxidation of diesel fuel vapors may be a good proxy for SOA from anthropogenic emissions. In our experiments, LLPS occurred over the relative humidity (RH) range of ~ 70 % to ~ 100 %, resulting in an organic-rich outer phase and a water-rich inner phase. These results may have implications for predicting the cloud nucleating properties of anthropogenic SOA since the organic-rich outer phase can lower the kinetic barrier for activation to a cloud droplet. At &leq; 10 % RH, the viscosity was in the range of &geq; 1 × 108 Pa s, which corresponds to roughly the viscosity of tar pitch. At 38–50 % RH the viscosity was in the range of 1 × 108–3 × 105 Pa s. These measured viscosities are consistent with predictions based on oxygen to carbon elemental ratio (O : C) and molar mass as well as predictions based on the number of carbon, hydrogen, and oxygen atoms. Based on the measured viscosities and the Stokes–Einstein relation, at &leq; 10 % RH diffusion coefficients of organics within diesel fuel SOA is &leq; 5.4 × 10−17cm2 s−1 and the mixing time of organics within 200 nm diesel fuel SOA particles (τmixing) is &gtrsim; 50 h. These small diffusion coefficients and large mixing times may be important in laboratory experiments, where SOA is often generated and studied using low RH conditions and on time scales of minutes to hours. At 38–50 % RH, the calculated organic diffusion coefficients are in the range of 5.4 × 10−17 to 1.8 × 10−13 cm2 s−1 and calculated τmixing values are in the range of ~ 0.01 h to ~ 50 h. These values provide important constraints for the physicochemical properties of anthropogenic SOA.

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Liquid–liquid phase separation and viscosity within secondary organic aerosol generated from diesel fuel vapors

Atmospheric Chemistry and Physics ◽

10.5194/acp-19-12515-2019 ◽

2019 ◽

Vol 19 (19) ◽

pp. 12515-12529 ◽

Cited By ~ 5

Author(s):

Mijung Song ◽

Adrian M. Maclean ◽

Yuanzhou Huang ◽

Natalie R. Smith ◽

Sandra L. Blair ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Diesel Fuel ◽

Diffusion Coefficients ◽

Secondary Organic Aerosol ◽

Organic Aerosol ◽

Liquid Phase Separation ◽

Small Diffusion ◽

Wide Range ◽

Liquid Liquid Phase Separation

Abstract. Information on liquid–liquid phase separation (LLPS) and viscosity (or diffusion) within secondary organic aerosol (SOA) is needed to improve predictions of particle size, mass, reactivity, and cloud nucleating properties in the atmosphere. Here we report on LLPS and viscosities within SOA generated by the photooxidation of diesel fuel vapors. Diesel fuel contains a wide range of volatile organic compounds, and SOA generated by the photooxidation of diesel fuel vapors may be a good proxy for SOA from anthropogenic emissions. In our experiments, LLPS occurred over the relative humidity (RH) range of ∼70 % to ∼100 %, resulting in an organic-rich outer phase and a water-rich inner phase. These results may have implications for predicting the cloud nucleating properties of anthropogenic SOA since the presence of an organic-rich outer phase at high-RH values can lower the supersaturation with respect to water required for cloud droplet formation. At ≤10 % RH, the viscosity was ≥1×108 Pa s, which corresponds to roughly the viscosity of tar pitch. At 38 %–50 % RH, the viscosity was in the range of 1×108 to 3×105 Pa s. These measured viscosities are consistent with predictions based on oxygen to carbon elemental ratio (O:C) and molar mass as well as predictions based on the number of carbon, hydrogen, and oxygen atoms. Based on the measured viscosities and the Stokes–Einstein relation, at ≤10 % RH diffusion coefficients of organics within diesel fuel SOA is ≤5.4×10-17 cm2 s−1 and the mixing time of organics within 200 nm diesel fuel SOA particles (τmixing) is 50 h. These small diffusion coefficients and large mixing times may be important in laboratory experiments, where SOA is often generated and studied using low-RH conditions and on timescales of minutes to hours. At 38 %–50 % RH, the calculated organic diffusion coefficients are in the range of 5.4×10-17 to 1.8×10-13 cm2 s−1 and calculated τmixing values are in the range of ∼0.01 h to ∼50 h. These values provide important constraints for the physicochemical properties of anthropogenic SOA.

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Charge-Driven Condensation of RNA and Proteins Suggests Broad Role of Phase Separation in Cytoplasmic Environments

10.1101/2020.04.23.057901 ◽

2020 ◽

Author(s):

Bercem Dutagaci ◽

Grzegorz Nawrocki ◽

Joyce Goodluck ◽

Ali Akbar Ashkarran ◽

Charles G. Hoogstraten ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Molecular Size ◽

Liquid Phase Separation ◽

Coarse Grained ◽

Biological Macromolecules ◽

Separation Processes ◽

Cellular Environment ◽

Liquid Liquid Phase Separation

ABSTRACTPhase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well established drivers of liquid-liquid phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Condensates retain liquid character and phase diagrams are mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components.STATEMENT OF SIGNIFICANCELiquid-liquid phase separation has been recognized as a key mechanism for forming membrane-less organelles in cells. Commonly discussed mechanisms invoke a role of disordered peptides and specific multi-valent interactions. We report here phase separation of RNA and proteins based on a more universal principle of charge complementarity that does not require disorder or specific interactions. The findings are supported by coarse-grained simulations, theory, and experimental validation via microscopy and spectroscopy. The broad implication of this work is that condensate formation may be a universal phenomenon in biological systems.

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Three archetypical classes of macromolecular regulators of protein liquid–liquid phase separation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907849116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19474-19483 ◽

Cited By ~ 22

Author(s):

Archishman Ghosh ◽

Konstantinos Mazarakos ◽

Huan-Xiang Zhou

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Computational Study ◽

Quantitative Measure ◽

Liquid Phase Separation ◽

Wide Range ◽

Regulatory Effects ◽

High Concentrations ◽

Droplet Phase ◽

Liquid Liquid Phase Separation

Membraneless organelles, corresponding to the droplet phase upon liquid–liquid phase separation (LLPS) of protein or protein–RNA mixtures, mediate myriad cellular functions. Cells use a variety of biochemical signals such as expression level and posttranslational modification to regulate droplet formation and dissolution, but the physical basis of the regulatory mechanisms remains ill-defined and quantitative assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromolecular regulators promote or suppress LLPS. We experimentally tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH35 and PRM5) as droplet-forming proteins. Determination of the changes in phase boundary and the partition coefficients in the droplet phase over a wide range of regulator concentrations yielded both a quantitative measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were observed. Ficoll 70 at high concentrations indirectly promoted SH35–PRM5 LLPS, by taking up volume in the bulk phase and thereby displacing SH35 and PRM5 into the droplet phase. Lysozyme had a moderate partition coefficient and suppressed LLPS by substituting weaker attraction with SH35 for the stronger SH35–PRM5 attraction in the droplet phase. By forming even stronger attraction with PRM5, heparin at low concentrations partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromolecular regulators as volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.

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Water uptake of subpollen aerosol particles: hygroscopic growth, CCN activation, and liquid-liquid phase separation

10.5194/acp-2020-1224 ◽

2020 ◽

Author(s):

Eugene F. Mikhailov ◽

Mira L. Pöhlker ◽

Kathrin Reinmuth-Selzle ◽

Sergey S. Vlasenko ◽

Ovid O. Krüger ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Water Uptake ◽

Chemical Reactivity ◽

Fine Fraction ◽

Liquid Phase Separation ◽

Hygroscopic Growth ◽

Model Calculations ◽

Wide Range ◽

Liquid Liquid Phase Separation

Abstract. Pollen grains emitted from vegetation can release subpollen particles (SPP) that contribute to the fine fraction of atmospheric aerosols and may act as cloud condensation nuclei (CCN), ice nuclei (IN), or aeroallergens. Here, we investigate and characterize the hygroscopic growth and CCN activation of birch, pine, and rapeseed SPP. A high humidity tandem differential mobility analyzer (HHTDMA) was used to measure particle restructuring and water uptake over a wide range of relative humidity (RH) from 2 % to 99.5 %, and a continuous flow CCN counter was used for size-resolved measurements of CCN activation at supersaturations (S) in the range of 0.2 % to 1.2 %. For both, subsaturated and supersaturated conditions, effective hygroscopicity parameters κ, were obtained by Köhler model calculations. Gravimetric and chemical analyses, electron microscopy, and dynamic light scattering measurements were performed to characterize further properties of SPP from aqueous pollen extracts such as chemical composition (starch, proteins, DNA, and inorganic ions) and the hydrodynamic size distribution of water-insoluble material. All investigated SPP samples exhibited sharp increases of water uptake and κ above ~95 % RH, suggesting a liquid-liquid phase separation (LLPS). The HHTDMA measurements at RH > 95 % enable closure between the CCN activation at water vapor supersaturation and hygroscopic growth at subsaturated conditions, which is often not achieved when HTDMA measurements are performed at lower RH where the water uptake and effective hygroscopicity may be limited by the effects of LLPS. Such effects may be important not only for closure between hygroscopic growth and CCN activation but also for the chemical reactivity, allergenic potential, and related health effects of SPP.

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CRISPR-based DNA and RNA detection with liquid-liquid phase separation

10.1101/471482 ◽

2018 ◽

Cited By ~ 3

Author(s):

Willem Kasper Spoelstra ◽

Jeroen M. Jacques ◽

Franklin L. Nobrega ◽

Anna C. Haagsma ◽

Marileen Dogterom ◽

...

Keyword(s):

Phase Separation ◽

Nucleic Acid ◽

Liquid Phase ◽

Clinical Diagnostics ◽

Liquid Phase Separation ◽

Molecular Diagnostic ◽

Target Sequence ◽

Dna And Rna ◽

Wide Range ◽

Liquid Liquid Phase Separation

AbstractThe ability to detect specific nucleic acid sequences allows for a wide range of applications including identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence both enzymes activate a unique, indiscriminate nucleic acid cleavage activity, which has been exploited for detection of sequence specific nucleotides using labelled reporter molecules. We here present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Turbidity arises from coacervates of positively charged polyelectrolytes with long poly(dT) or poly(U) oligonucleotides. In the presence of a target sequence, long oligonucleotides are progressively shortened, changing the solution from turbid to transparent. We explain how oligonucleotide cleavage resolves LLPS by using a mathematical model which we validate with poly(dT) phase separation experiments. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.

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Structure of biomolecular condensates from dissipative particle dynamics simulations

10.1101/2019.12.11.873133 ◽

2019 ◽

Author(s):

Julian C. Shillcock ◽

Maelick Brochut ◽

Etienne Chénais ◽

John H. Ipsen

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Intrinsically Disordered Proteins ◽

Liquid Phase Separation ◽

Coarse Grained ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Wide Range ◽

End Caps ◽

Liquid Liquid Phase Separation

ABSTRACTPhase separation of immiscible fluids is a common phenomenon in polymer chemistry, and is recognized as an important mechanism by which cells compartmentalize their biochemical reactions. Biomolecular condensates are condensed fluid droplets in cells that form by liquid-liquid phase separation of intrinsically-disordered proteins. They have a wide range of functions and are associated with chronic neurodegenerative diseases in which they become pathologically rigid. Intrinsically-disordered proteins are conformationally flexible and possess multiple, distributed binding sites for each other or for RNA. However, it remains unclear how their material properties depend on the molecular structure of the proteins. Here we use coarse-grained simulations to explore the phase behavior and structure of a model biomolecular condensate composed of semi-flexible polymers with attractive end-caps in a good solvent. Although highly simplified, the model contains the minimal molecular features that are sufficient to observe liquid-liquid phase separation of soluble polymers. The polymers condense into a porous, three-dimensional network in which their end-caps reversibly bind at junctions. The spatial separation of connected junctions scales with the polymer backbone length as a self-avoiding random walk over a wide range of concentration with a weak affinity-dependent prefactor. By contrast, the average number of polymers that meet at the junctions depends strongly on the end-cap affinity but only weakly on the polymer length. The regularity and porosity of the condensed network suggests a mechanism for cells to regulate biomolecular condensates. Interaction sites along a protein may be turned on or off to modulate the condensate’s porosity and tune the diffusion and interaction of additional proteins.

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Self-limited uptake of α-pinene-oxide to acidic aerosol: the effects of liquid-liquid phase separation and implications for the formation of secondary organic aerosol and organosulfates from epoxides

Atmospheric Chemistry and Physics Discussions ◽

10.5194/acpd-13-7151-2013 ◽

2013 ◽

Vol 13 (3) ◽

pp. 7151-7174

Author(s):

G. T. Drozd ◽

J. L. Woo ◽

V. F. McNeill

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Partition Coefficients ◽

Secondary Organic Aerosol ◽

Organic Aerosol ◽

Organic Content ◽

Liquid Phase Separation ◽

Wide Range ◽

Acidic Sulfate ◽

Liquid Liquid Phase Separation

Abstract. The reactive uptake of α-pinene oxide (αPO) to acidic sulfate aerosol was studied under humid conditions in order to gain insight into the effects of liquid-liquid phase separation on aerosol heterogeneous chemistry and further elucidate the formation of secondary organic aerosol and organosulfates from epoxides. A continuous flow environmental chamber was used to monitor changes in diameter of monodisperse, deliquesced, acidic sulfate particles exposed to αPO at 30 and 50% RH. In order to induce phase separation and probe potential limits to particle growth from acidic uptake, αPO was introduced over a wide range of concentrations, from 200 ppb to 5 ppm. Uptake was observed to be highly dependent on initial aerosol pH. Significant uptake of αPO to aerosol was observed with initial pH < 0. When exposed to 200 ppb αPO, aerosol with pH = −1 doubled in volume, and 6% volume growth was observed at pH = 0. Aerosol with pH = 1 showed no growth. The extreme acidity required for efficient αPO uptake suggests that this chemistry is typically not a major route to formation of aerosol mass or organosulfates in the atmosphere. Partition coefficients (Kp) ranged from 0.2–1.6 × 10−4 m3 μg−1 and were correlated to initial particle acidity and particle organic content; particles with higher organic content had lower partition coefficients. Effective uptake coefficients (γ) ranged from 0.4 to 4.7 × 10−5 and are much lower than recently reported for uptake to bulk solutions. In experiments in which αPO was added to bulk H2SO4 solutions, phase separation was observed for mass loadings similar to those observed with particles, and product distributions were dependent on acid concentration. Liquid-liquid phase separation in bulk experiments, along with our observations of decreased uptake to particles with the largest growth factors, suggest an organic coating forms upon uptake to particles, limiting reactive uptake.

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