Properties Of Condensate Research Articles

Sequence-specific insights into the time-dependent material properties of biomolecular condensates

Sequence-specific insights into the time-dependent material properties of biomolecular condensates

Temperature-controlled microrheology illuminates distinctive roles of chain length and sticker strength on material properties of biomolecular condensates

Temperature-controlled microrheology illuminates distinctive roles of chain length and sticker strength on material properties of biomolecular condensates

Quantification and regulation of the material properties of synapsin condensates in vitro and in live cells

Quantification and regulation of the material properties of synapsin condensates in vitro and in live cells

Active learning of the thermodynamics-dynamics trade-off in protein condensates.

Phase-separated biomolecular condensates exhibit a wide range of dynamic properties, which depend on the sequences of the constituent proteins and RNAs. However, it is unclear to what extent condensate dynamics can be tuned without also changing the thermodynamic properties that govern phase separation. Using coarse-grained simulations of intrinsically disordered proteins, we show that the dynamics and thermodynamics of homopolymer condensates are strongly correlated, with increased condensate stability being coincident with low mobilities and high viscosities. We then apply an "active learning" strategy to identify heteropolymer sequences that break this correlation. This data-driven approach and accompanying analysis reveal how heterogeneous amino acid compositions and nonuniform sequence patterning map to a range of independently tunable dynamic and thermodynamic properties of biomolecular condensates. Our results highlight key molecular determinants governing the physical properties of biomolecular condensates and establish design rules for the development of stimuli-responsive biomaterials.

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

Recombinant silk protein condensates show widely different properties depending on the sample background.

There is an increasing understanding that condensation is a crucial intermediate step in the assembly of biological materials and for a multitude of cellular processes. To apply and to understand these mechanisms, in vitro biophysical characterisation techniques are central. The formation and biophysical properties of protein condensates depend on a multitude of factors, such as protein concentration, pH, temperature, salt concentration, and presence of other biomolecules as well as protein purification and storage conditions. Here we show how critical the procedures for preparing protein samples for in vitro studies are. We compare two purification methods of the recombinant spider silk protein CBM-AQ12-CBM and study the effect of background molecules, such as DNA, on the formation and properties of the condensates. We characterize the condensates using aggregation induced emitters (AIEs), coalescence studies, and micropipette aspiration. The condensated sample containing background molecules exhibit a lower threshold concentration for condensate formation accompanied by a lower surface tension and longer coalescence time when compared to the pure protein condensates. Furthermore, the partitioning of small AIEs is enhanced in the presence of background molecules. Our results highlight that the purification method and remaining background molecules strongly affect the biophysical properties of spider silk condensates. Using the acquired knowledge about spider silk protein purification we derive guidelines for reproducible condensate formation that will foster the use of spider silk proteins as adhesives or carriers for biomedical applications.

Effects of charge asymmetry on the liquid-liquid phase separation of polyampholytes and their condensate properties.

Liquid-liquid phase separation (LLPS) is the mechanism underlying the formation of bio-molecular condensates which are important compartments regulating intra- and extra-cellular functions. Electrostatic interactions are some of the important driving forces of the LLPS behaviors of biomolecules. However, the understanding of the electrostatic interactions is still limited, especially in the mixtures of biomolecules with different charge patterns. Here, we focus on the electrostatic interactions in mixtures of charge-asymmetric and charge-symmetric polyampholytes and their roles in the phase separation behaviors. We build charge-asymmetric and charge-symmetric model proteins consisting of both glutamic acid (E, negatively charged) and lysine (K, positively charged), i.e. polyampholytes of E35K15 (charge asymmetric) and E25K25 (charge symmetric). Pure E25K25 can undergo LLPS. To investigate the effects of charge-asymmetric polyampholytes on the mixtures of E25K25/E35K15, we perform coarse-grained simulations to determine their phase separation. The charge-asymmetric polyampholyte E35K15 is resistant to the LLPS of the mixtures of E25K25/E35K15. The condensate density decreases with the molar fraction of E35K15 increasing to 0.4, and no LLPS occurs at the molar fraction of 0.5 and above. This can be attributed to the electrostatic repulsion between the negatively charged E35K15 polymers. We further investigate the effects of charge asymmetry on the conformations and properties of the condensates. The E35K15 polymers in the condensates exhibit a more collapsed state as the molar fraction of E35K15 increases. However, the conformation of E25K25 polymers changes slightly across different condensates. The surface tensions of condensates decline with the increase of the molar fraction of E35K15 polymers, while the diffusivity of polymers in the condensed phases is enhanced. This work elucidates the role of charge-asymmetric polyampholytes in determining the LLPS behaviours of binary mixtures of charge-symmetric and charge-asymmetric proteins as well as the properties of condensed phases.

Lipidation alters the phase-separation of resilin-like polypeptides.

Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)-to modulate the amino acid physicochemistry instead of altering protein sequences-to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation-in synergy with the sequence of the lipidation site-significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of "sequence context" in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.

A chaperone-like function of FUS ensures TAZ condensate dynamics and transcriptional activation.

The Hippo pathway has important roles in organ development, tissue homeostasis and tumour growth. Its downstream effector TAZ is a transcriptional coactivator that promotes target gene expression through the formation of biomolecular condensates. However, the mechanisms that regulate the biophysical properties of TAZ condensates to enable Hippo signalling are not well understood. Here using chemical crosslinking combined with an unbiased proteomics approach, we show that FUS associates with TAZ condensates and exerts a chaperone-like effect to maintain their proper liquidity and robust transcriptional activity. Mechanistically, the low complexity sequence domain of FUS targets the coiled-coil domain of TAZ in a phosphorylation-regulated manner, which ensures the liquidity and dynamicity of TAZ condensates. In cells lacking FUS, TAZ condensates transition into gel-like or solid-like assembles with immobilized TAZ, which leads to reduced expression of target genes and inhibition of pro-tumorigenic activity. Thus, our findings identify a chaperone-like function of FUS in Hippo regulation and demonstrate that appropriate biophysical properties of transcriptional condensates are essential for gene activation.

Dipping contacts - a novel type of contact site at the interface between membraneless organelles and membranes.

Liquid-liquid phase separation is a major mechanism for organizing macromolecules, particularly proteins with intrinsically disordered regions, in compartments not limited by a membrane or a scaffold. The cell can therefore be perceived as a complex emulsion containing many of these membraneless organelles, also referred to as biomolecular condensates, together with numerous membrane-bound organelles. It is currently unclear how such a complex concoction operates to allow for intracellular trafficking, signaling and metabolic processes to occur with high spatiotemporal precision. Based on experimental observations of synaptic vesicle condensates - a membraneless organelle that is in fact packed with membranes - we present here the framework of dipping contacts: a novel type of contact site between membraneless organelles and membranes. In this Hypothesis, we propose that our framework of dipping contacts can serve as a foundation to investigate the interface that couples the diffusion and material properties of condensates to biochemical processes occurring in membranes. The identity and regulation of this interface is especially critical in the case of neurodegenerative diseases, where aberrant inclusions of misfolded proteins and damaged organelles underlie cellular pathology.

Droplet Physics and Intracellular Phase Separation

Living cells are spatially organized by compartments that can nucleate, grow, and dissolve. Compartmentalization can emerge by phase separation, leading to the formation of droplets in the cell's nucleo- or cytoplasm, also called biomolecular condensates. Such droplets can organize the biochemistry of the cell by providing specific chemical environments in space and time. These compartments provide transient environments, suggesting the relevance of nonequilibrium physics of droplets as a key to unraveling the underlying physicochemical principles of biological functions in living cells. In this review, we highlight coarse-grained approaches that capture the physics of chemically active emulsions as a model for condensates orchestrating chemical processes. We also discuss the dynamics of single molecules in condensates and the material properties of biological condensates and their relevance for the cell. Finally, we propose wetting, prewetting, and surface phase transitions as a possibility for intracellular surfaces to control biological condensates, spatially organize membranes, and exert mechanical forces.

Photoelectric properties of thin layers of Ag2S quantum dots passivated with thiol-containing molecules

In this work, we studied the electrophysical properties of condensates of colloidal Ag2S quantum dots (QDs), passivated with thiol-containing molecules (thioglycolic acid and L-cysteine) (further, Ag2S/TGA QDs and Ag2S/L-Cys QDs) on indium tin oxide (ITO) substrates. The temperature dependences of the electrical characteristics (conductivity) are analyzed in the temperature range from 300 to 360 K. Facts of the formation of the Schottky barrier structures is obtained. Activation energies of 0.2 eV (Ag2S/L-Cys QDs) and 0.66 eV (Ag2S/TGA QDs) were obtained by the use of linear approximations of current–voltage (I–V) characteristics in Arrhenius coordinates. It is shown that the conductivity of ITO - Ag2S QDs film - Al structure is mainly determined by the Schottky barrier that is formed at the Al-Ag2S QDs film junction. The rectifying contact is formed at the Ag2S film -Al junction due to the lower work function of Al. The I–V characteristics under the optical radiation action were analyzed for the studied sandwich structures. They turned out to be similar for Ag2S/TGA and Ag2S/L-Cys QDs. When a reverse or forward bias voltage is applied to a sandwich structure, in the case of the optical radiation action with a wavelength starting from the its value, corresponding to the most probable exciton transition in the QDs absorption the photocurrent is observed. The found difference (by an order of magnitude) in the photocurrent values of the sandwich structure, based on Ag2S/TGA QDs and Ag2S/L-Cys QDs is related to the smaller thickness of the Ag2S/TGA QDs film.

Spatial Imaging and Control of Dark Excitons in Monolayer Transition Metal Dichalcogenides.

Dark excitons play a vital role in exciton condensation and optical properties of monolayer transition metal dichalcogenides (MTMDs). Previous literature mainly focuses on the detection of the energy of the dark exciton, while spatial detection and control are equally important but are less studied. Here we report that for MTMD embedded in a semiconductor microcavity and under a uniform in-plane magnetic field the spatial distribution of the dark exciton can be probed by measuring that of the cavity photon for small exciton-exciton interaction energy. Further, we propose to realize the anomalous exciton Hall effect by exploiting spatially inhomogeneous coupling of the bright and dark excitons under a Gaussian excitation beam. This effect occurs regardless of the exciton-exciton interaction, which will strengthen the diffusion of excitons in the excitation region. These results provide an improved understanding of the excitons in MTMDs, thereby facilitating their potential practical applications.

Triblock Proteins with Weakly Dimerizing Terminal Blocks and an Intrinsically Disordered Region for Rational Design of Condensate Properties.

Condensates are molecular assemblies that are formed through liquid-liquid phase separation and play important roles in many biological processes. The rational design of condensate formation and their properties is central to applications, such as biosynthetic materials, synthetic biology, and for understanding cell biology. Protein engineering is used to make a triblock structure with varying terminal blocks of folded proteins on both sides of an intrinsically disordered mid-region. Dissociation constants are determined in the range of micromolar to millimolar for a set of proteins suitable for use as terminal blocks. Varying the weak dimerization of terminal blocks leads to an adjustable tendency for condensate formation while keeping the intrinsically disordered region constant. The dissociation constants of the terminal domains correlate directly with the tendency to undergo liquid-liquid phase separation. Differences in physical properties, such as diffusion rate are not directly correlated with the strength of dimerization but can be understood from the properties and interplay of the constituent blocks. The work demonstrates the importance of weak interactions in condensate formation and shows a principle for protein design that will help in fabricating functional condensates in a predictable and rational way.

A novel carbon dioxide capture technology (CCT) based on non-equilibrium condensation characteristics: Numerical modelling, nozzle design and structure optimization

The carbon capture technology (CCT) is widely studied to decrease carbon emissions and improve the global climate. Supersonic separation is becoming a new strategy for decarbonization. In order to simulate the non-equilibrium condensing flow of CO2 in the supersonic nozzle more accurately, the mathematical model is modified and verified, and non-equilibrium condensation of carbon dioxide in different states and different inlet superheat levels are analysed numerically. In order to improve the separation efficiency of the supersonic decarbonization technology and reduce the energy loss, the nozzle structure is appropriately designed and optimized. Firstly, the model is verified by gas properties, different droplet growth models and surface tension models. The result shows that the real gas model can predict the non-equilibrium condensing flow in the nozzle more accurately than the ideal gas model. Among the four droplet growth models, the most suitable one was selected in order to predict the non-equilibrium condensation properties of CO2. The droplet surface tension is corrected by introducing temperature and the droplet radius, and the droplet condensation process is enhanced. Secondly, the condensing flow through the nozzle is predicted in different states (subcritical, near-critical and supercritical). The result shows that the prediction based on the modified model is basically consistent with experimental data. Analysing the predicted Wilson line, it is found that the non-equilibrium effect of the flow is quite different at different inlet conditions. The condensation locations gradually approach the nozzle throat, the condensation interval narrows with an increase in pressure, and the peak of the nucleation rate increases gradually. Furthermore, the effect of inlet superheat on the non-equilibrium condensing flow is investigated. As the temperature rises, the flow loss decreases and the thermal efficiency increases from 61.15% to 67.62%. Finally, four nozzles with different convergence shapes are calculated. One of them demonstrates stronger stability, lower losses and the highest thermal efficiency which is about 67.64%. Additionally, when the expansion angle is 7°, the average of the liquid mass fraction is the highest, about 21.7%.

Superior TRAIL gene expression and cancer cell apoptosis mediated by highly branched-linear poly(β-amino ester)s

Extensive efforts have been dedicated to enhancing the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in cancer cells for the development of effective cancer treatments. However, highly safe and efficient delivery of TRAIL gene remains a significant challenge, especially using cationic polymers. Here, a series of highly branched-linear poly(β-amino ester)s (H-LPAEs) are developed through a unique oligomer branching strategy. H-LPAEs exhibit a more uniform distribution of linear segments and branching units, leading to excellent DNA condensation and favorable physicochemical properties of H-LPAE/DNA polyplexes. In SW1353 and BMSC cells, the optimized H-LPAEs, H-LPAEB4−S5−TMPTA, achieves superior gene transfection efficiency of 58.0% and 33.4%, which were 2.5-fold and 2.0-fold higher than that of the leading commercial gene transfection reagent, Lipofectamine 3000. Excitingly, H-LPAEB4−S5−TMPTA mediated 56.7% and 28.1% cell apoptosis in HepG2 cells and HeLa cells highlighting its potential application in cancer gene therapy. In addition, locally administered H-LPAEB4−S5−TMPTA delivered TRAIL DNA to HepG2 xenograft tumors and inhibited tumor growth in vivo. This study not only proposes a novel strategy for synthesizing poly(β-amino ester)s with a unique branched-linear topology but also identifies a promising candidate for highly efficient TRAIL gene transfection.

Effect of ethane on condensation characteristics of methane heterogeneous nucleation: A molecular dynamics study

Clarification on condensation characteristics of alkane gases plays a fundamental role in the development of natural gas liquefaction processes. However, the microscopic condensation characteristics of alkane gas on the heat exchanger wall are still not well understood, which holds significant practical implications for advancing liquefaction technologies. Therefore, this study investigates the microscopic condensation process of heterogeneous nucleation with methane gas through molecular dynamics (MD) simulations. By varying wall energy and ethane content, we analyzed the nucleation kinetic properties of methane heterogeneous condensation. The results demonstrate that high-energy walls enhance heat transfer by enhancing the interaction between wall and methane, thereby accelerating condensation core formation and increasing methane condensation rates. Additionally, increasing ethane content also shortens the time for the formation of condensation cores in methane gas. Ethane has a higher condensation temperature compared to methane, and its presence reduces the difficulty of forming condensation cores in alkane gases and shortens the time for liquid core formation, thus facilitating methane condensation. The above research results will provide new insight for natural gas liquefaction process.

Conformational Properties of Polymers at Droplet Interfaces as Model Systems for Disordered Proteins.

Polymer models serve as useful tools for studying the formation and physical properties of biomolecular condensates. In recent years, the interface dividing the dense and dilute phases of condensates has been discovered to be closely related to their functionality, but the conformational preferences of the constituent proteins remain unclear. To elucidate this, we perform molecular simulations of a droplet formed by phase separation of homopolymers as a surrogate model for the prion-like low-complexity domains. By systematically analyzing the polymer conformations at different locations in the droplet, we find that the chains become compact at the droplet interface compared with the droplet interior. Further, segmental analysis revealed that the end sections of the chains are enriched at the interface to maximize conformational entropy and are more expanded than the middle sections of the chains. We find that the majority of chain segments lie tangential to the droplet surface, and only the chain ends tend to align perpendicular to the interface. These trends also hold for the natural proteins FUS LC and LAF-1 RGG, which exhibit more compact chain conformations at the interface compared to the droplet interior. Our findings provide important insights into the interfacial properties of biomolecular condensates and highlight the value of using simple polymer physics models to understand the underlying mechanisms.

A MEC-2/stomatin condensate liquid-to-solid phase transition controls neuronal mechanotransduction during touch sensing.

A growing body of work suggests that the material properties of biomolecular condensates ensuing from liquid-liquid phase separation change with time. How this aging process is controlled and whether the condensates with distinct material properties can have different biological functions is currently unknown. Using Caenorhabditis elegans as a model, we show that MEC-2/stomatin undergoes a rigidity phase transition from fluid-like to solid-like condensates that facilitate transport and mechanotransduction, respectively. This switch is triggered by the interaction between the SH3 domain of UNC-89 (titin/obscurin) and MEC-2. We suggest that this rigidity phase transition has a physiological role in frequency-dependent force transmission in mechanosensitive neurons during body wall touch. Our data demonstrate a function for the liquid and solid phases of MEC-2/stomatin condensates in facilitating transport or mechanotransduction, and a previously unidentified role for titin homologues in neurons.

Micromachining on Stainless Steel 304 for Improved Water Condensation Properties

Microstructure fabrication and chemical surface functionalization with low-surface-energy materials are the key steps to achieve hydrophobic surfaces with high water droplet contact angles (CA). In this work we employed wire Electric Discharge Machining (EDM) as a way to induce microstructure topography on stainless steel 304 coupons. The resulting topography was rendered hydrophobic using trichloro-1H,1H,2H,2H-perfluorooctyl silane (PFOTS) via gas phase deposition. The channels created by machining and PFOTS functionalization facilitate water condensation by increasing nucleation sites and enhancing droplet coalescence. The resulting surface is hydrophobic (CA~140o) in contrast to the bare stainless steel 304, which is hydrophilic (CA~76o).