A DNA condensation code for linker histones.

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.

Driving force and pathway in polyelectrolyte complex coacervation.

There is notable discrepancy between experiments and coarse-grained model studies regarding the thermodynamic driving force in polyelectrolyte complex coacervation: experiments find the free energy change to be dominated by entropy, while simulations using coarse-grained models with implicit solvent usually report a large, even dominant energetic contribution in systems with weak to intermediate electrostatic strength. Here, using coarse-grained, implicit-solvent molecular dynamics simulation combined with thermodynamic analysis, we study the potential of mean force (PMF) in the two key stages on the coacervation pathway for symmetric polyelectrolyte mixtures: polycation-polyanion complexation and polyion pair-pair condensation. We show that the temperature dependence in the dielectric constant of water gives rise to a substantial entropic contribution in the electrostatic interaction. By accounting for this electrostatic entropy, which is due to solvent reorganization, we find that under common conditions (monovalent ions, room temperature) for aqueous systems, both stages are strongly entropy-driven with negligible or even unfavorable energetic contributions, consistent with experimental results. Furthermore, for weak to intermediate electrostatic strengths, this electrostatic entropy, rather than the counterion-release entropy, is the primary entropy contribution. From the calculated PMF, we find that the supernatant phase consists predominantly of polyion pairs with vanishingly small concentration of bare polyelectrolytes, and we provide an estimate of the spinodal of the supernatant phase. Finally, we show that prior to contact, two neutral polyion pairs weakly attract each other by mutually induced polarization, providing the initial driving force for the fusion of the pairs.

Functional enzyme-polymer complexes.

SignificanceThe use of biological enzyme catalysts could have huge ramifications for chemical industries. However, these enzymes are often inactive in nonbiological conditions, such as high temperatures, present in industrial settings. Here, we show that the enzyme PETase (polyethylene terephthalate [PET]), with potential application in plastic recycling, is stabilized at elevated temperature through complexation with random copolymers. We demonstrate this through simulations and experiments on different types of substrates. Our simulations also provide strategies for designing more enzymatically active complexes by altering polymer composition and enzyme charge distribution.

Reflectivity and Angular Anisotropy of Liquid Crystal Microcapsules with Different Particle Sizes by Complex Coalescence.

Cholesteric liquid crystal microcapsules (CLCMs) are used to improve the stability of liquid crystals while ensuring their stimulus response performance and versatility, with representative applications such as sensing, anticounterfeiting, and smart fabrics. However, the reflectivity and angular anisotropy decrease because of the anchoring effect of the polymer shell matrix, and the influence of particle size on this has not been thoroughly studied. In this study, the effect of synthesis technology on microcapsule particle size was investigated using a complex coalescence method, and the effect of particle size on the reflectivity and angular anisotropy of CLCMs was investigated in detail. A particle size of approximately 66 µm with polyvinyl alcohol (PVA, 1:1) exhibited a relative reflectivity of 16.6% and a bandwidth of 20 nm, as well as a narrow particle size distribution of 22 µm. The thermosetting of microcapsules coated with PVA was adjusted and systematically investigated by controlling the mass ratio. The optimized mass ratio of microcapsules (66 µm) to PVA was 2:1, increasing the relative reflectivity from 16.6% (1:1) to 32.0% (2:1) because of both the higher CLCM content and the matching between the birefringence of the gelatin-arabic shell system and PVA. Furthermore, color based on Bragg reflections was observed in the CLCM-coated ortho-axis and blue-shifted off-axis, and this change was correlated with the CLCM particle size. Such materials are promising for anticounterfeiting and color-based applications with bright colors and angular anisotropy in reflection.

Microencapsulation of Essential Oils Using Faba Bean Protein and Chia Seed Polysaccharides via Complex Coacervation Method.

The aim of this study was to develop microcapsules containing juniper or black pepper essential oils, using a combination of faba bean protein and chia seed polysaccharides (in ratios of 1:1, 1:2, 2:1). By synergizing these two polymers, our goal was to enhance the efficiency of essential oil microencapsulation, opening up various applications in the food industry. Additionally, we aimed to investigate the influence of different polymer mixing ratios on the properties of the resulting microcapsules and the course of the complex coacervation process. To dissolve the essential oils and limit their evaporation, soybean and rapeseed oils were used. The powders resulting from the freeze-drying of coacervates underwent testing to assess microencapsulation efficiency (65.64-87.85%), density, flowability, water content, solubility, and hygroscopicity. Additionally, FT-IR and DSC analyses were conducted. FT-IR analysis confirmed the interactions between the components of the microcapsules, and these interactions were reflected in their high thermal resistance, especially at a protein-to-polysaccharide ratio of 2:1 (177.2 °C). The water content in the obtained powders was low (3.72-7.65%), but it contributed to their hygroscopicity (40.40-76.98%).

Fuel-Driven Dynamic Combinatorial Peptide Libraries.

Dynamic combinatorial chemistry (DCC) creates libraries of molecules that are constantly interchanging in a dynamic combinatorial library. When a library member self-assembles, it can displace the equilibria, leading to emergent phenomena like its selection or even its replication. However, such dynamic combinatorial libraries typically operate in or close to equilibrium. This work introduces a new dynamic combinatorial chemistry fueled by a catalytic reaction cycle that forms transient, out-of-equilibrium peptide-based macrocycles. The products in this library exist out of equilibrium at the expense of fuel and are thus regulated by kinetics and thermodynamics. By creating a chemically fueled dynamic combinatorial library with the vast structural space of amino acids, we explored the liquid-liquid phase separation behavior of the library members. The study advances DCCs by showing that peptide structures can be engineered to control the dynamic library's behavior. The work paves the way for creating novel, tunable material systems that exhibit emergent behavior reminiscent of biological systems. These findings have implications for the development of new materials and for understanding life's chemistry.

Encapsulation of tannins and tannin-rich plant extracts by complex coacervation to improve their physicochemical properties and biological activities: A review.

As a major class of dietary polyphenols, tannins are demonstrated to have various health-promoting properties. Although tannins have been widely utilized in food, pharmaceutical and many other industries, the applications of tannins are quite limited due to their poor stability, sensory attributes and bioavailability. Encapsulation helps improve all of these properties. Complex coacervation, one of the most effective encapsulation techniques, is known for its simplicity, low cost, scalability and reproducibility in encapsulation of functional components. In recent years, complex coacervation has been successfully used for encapsulation of tannins and tannin-rich plant extracts. In this article, the research progress in encapsulating tannins and tannin-rich plant extracts by complex coacervation to improve their physicochemical properties and biological activities is critically reviewed for the first time. Encapsulation of tannins and tannin-rich plant extracts can effectively improve their sensory characteristics, stabilities, bioavailability, anti-hypercholesterolemia, anti-diabetic, antioxidant, anticancer and antimicrobial activities. In particular, the enhancement of biological activities of tannins and tannin-rich plant extracts is usually correlated to their improved physicochemical properties imparted by the encapsulation technique. Moreover, we introduce the issues that need to be further resolved in future studies on encapsulation of tannins and tannin-rich plant extracts by complex coacervation.

Improved Viability of Probiotics via Microencapsulation in Whey-Protein-Isolate-Octenyl-Succinic-Anhydride-Starch-Complex Coacervates.

The aim of this study was to microencapsulate probiotic bacteria (Lactobacillus acidophilus 11073) using whey-protein-isolate (WPI)-octenyl-succinic-anhydride-starch (OSA-starch)-complex coacervates and to investigate the effects on probiotic bacterial viability during spray drying, simulated gastrointestinal digestion, thermal treatment and long-term storage. The optimum mixing ratio and pH for the preparation of WPI-OSA-starch-complex coacervates were determined to be 2:1 and 4.0, respectively. The combination of WPI and OSA starch under these conditions produced microcapsules with smoother surfaces and more compact structures than WPI-OSA starch alone, due to the electrostatic attraction between WPI and OSA starch. As a result, WPI-OSA-starch microcapsules showed significantly (p < 0.05) higher viability (95.94 ± 1.64%) after spray drying and significantly (p < 0.05) better protection during simulated gastrointestinal digestion, heating (65 °C/30 min and 75 °C/10 min) and storage (4/25 °C for 12 weeks) than WPI-OSA-starch microcapsules. These results demonstrated that WPI-OSA-starch-complex coacervates have excellent potential as a novel wall material for probiotic microencapsulation.

Complex coacervation and freeze drying using whey protein concentrate, soy protein isolate and arabic gum to improve the oxidative stability of chia oil.

BACKGROUND: Chia oil (CO) is popular for being the richest vegetable source of α-linolenic acid (60-66%). However, this content of polyunsaturated fatty acids (PUFA) limits the incorporation of bulk CO in food products due to its high probability of oxidation. This justifies the study of alternative wall materials for microencapsulation. No reports regarding the use of dairy protein/vegetable protein/polysaccharide blends as wall material for the microencapsulation of CO have been published. Therefore, this work analyzed the behavior of a whey protein concentrate (WPC)/soy protein isolate (SPI)/arabic gum (AG) blend as wall material. The complex coacervation (CC) process was studied: pH, 4.0; total solid content, 30% w/v; WPC/SPI/AG ratio, 8:1:1 w/w/w; stirring speed, 600 rpm; time, 30 min; room temperature. RESULTS: The oxidative stability index (OSI) of CO (3.25 ± 0.16 h) was significantly increased after microencapsulation (around four times higher). Furthermore, the well-known matrix-forming ability of AG and WPC helped increase the OSI of microencapsulated oils. Meanwhile, SPI contributed to the increase of the encapsulation efficiency due to its high viscosity. Enhanced properties were observed with CC: encapsulation efficiency (up to 79.88%), OSIs (from 11.25 to 12.52 h) and thermal stability of microcapsules given by the denaturation peak temperatures of WPC (from 77.12 to 86.00 °C). No significant differences were observed in the fatty acid composition of bulk and microencapsulated oils. CONCLUSION: Microcapsules developed from complex coacervates based on the ternary blend represent promising omega-3-rich carriers for being incorporated into functional foods.

Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties.

Phase separation of multivalent protein and RNA molecules underlies the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that typically organize into multilayered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we deploy systematic experiments and simulations based on coarse-grained models to show that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multilayered condensates with distinct material properties. These studies yield a set of rules regarding homotypic and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.

Microencapsulation of Lactobacillus plantarum LN66 and its survival potential under different packaging conditions.

Microcapsules of Lactobacillus plantarum LN66 were prepared to improve the cell viability in simulated gastrointestinal and different packaging conditions. Microcapsules containing Lactobacillus plantarum LN66 were produced by complex coacervation followed by freeze drying and characterised by water activity, moisture content, size, encapsulation efficiency, SEM, FTIR, XRD, as well as the resistance of probiotics to the simulated gastrointestinal tract and storage under different packaging conditions. The microcapsules presented the particle size of 196.57 ± 1.46 µm and the encapsulation efficiency of 75.26 ± 1.95% (w/w). After simulated gastrointestinal conditions, viability of encapsulated cells was 71.33 ± 0.99% (w/w) and 70.39 ± 0.86% (w/w), separately, while that of free cells was only 45.45 ± 0.5% (w/w) and 8.59 ± 0.67% (w/w). Compared with aluminium foil, the viable cells in glass bottles at 4 °C and 25 °C was increased 1.1-fold and 1.4-fold, respectively. Complex coacervation could be considered an appropriate alternative to increase the viability of probiotics.

Coacervation as a Novel Method of Microencapsulation of Essential Oils-A Review.

These days, consumers are increasingly "nutritionally aware". The trend of "clean label" is gaining momentum. Synthetic additives and preservatives, as well as natural ones, bearing the E symbol are more often perceived negatively. For this reason, substances of natural origin are sought tfor replacing them. Essential oils can be such substances. However, the wider use of essential oils in the food industry is severely limited. This is because these substances are highly sensitive to light, oxygen, and temperature. This creates problems with their processing and storage. In addition, they have a strong smell and taste, which makes them unacceptable when added to the product. The solution to this situation seems to be microencapsulation through complex coacervation. To reduce the loss of essential oils and the undesirable chemical changes that may occur during their spray drying-the most commonly used method-complex coacervation seems to be an interesting alternative. This article collects information on the limitations of the use of essential oils in food and proposes a solution through complex coacervation with plant proteins and chia mucilage.

Encapsulation of Vitamin B12 by Complex Coacervation of Whey Protein Concentrate-Pectin; Optimization and Characterization.

Vitamin B12 (VB12) is one of the essential vitamins for the body, which is sensitive to light, heat, oxidizing agents, and acidic and alkaline substances. Therefore, the encapsulation of VB12 can be one of the ways to protect it against processing and environmental conditions in food. In this work, the influence of pectin concentration (0.5−1% w/v), whey protein concentrate (WPC) level (4−8% w/v) and pH (3−9) on some properties of VB12-loaded pectin−WPC complex carriers was investigated by response surface methodology (RSM). The findings showed that under optimum conditions (1:6.47, pectin:WPC and pH = 6.6), the encapsulation efficiency (EE), stability, viscosity, particle size and solubility of complex carriers were 80.71%, 85.38%, 39.58 mPa·s, 7.07 µm and 65.86%, respectively. Additionally, the formation of complex coacervate was confirmed by Fourier-transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). In addition, it was revealed that the most important factor in VB12 encapsulation was pH; at a pH < isoelectric point of WPC (pH = 3), in comparison with higher pH values (6 and 9), a stronger complex was formed between pectin and WPC, which led to an increase in EE, lightness parameter, particle size and water activity, as well as a decrease in the zeta-potential and porosity of complex carriers.

Phase Transitions in Chemically Fueled, Multiphase Complex Coacervate Droplets.

Membraneless organelles are droplets in the cytosol that are regulated by chemical reactions. Increasing studies suggest that they are internally organized. However, how these subcompartments are regulated remains elusive. Herein, we describe a complex coacervate-based model composed of two polyanions and a short peptide. With a chemical reaction cycle, we control the affinity of the peptide for the polyelectrolytes leading to distinct regimes inside the phase diagram. We study the transitions from one regime to another and identify new transitions that can only occur under kinetic control. Finally, we show that the chemical reaction cycle controls the liquidity of the droplets offering insights into how active processes inside cells play an important role in tuning the liquid state of membraneless organelles. Our work demonstrates that not only thermodynamic properties but also kinetics should be considered in the organization of multiple phases in droplets.

Phase Separation of Oppositely Charged Polymers Regulates Bioinspired Silicification.

In nature, simple organisms evolved mechanisms to form intricate biosilica nanostructures, far exceeding current synthetic manufacturing. Based on the properties of extracted biomacromolecules, polycation-polyanion pairs were suggested as moderators of biosilica formation. However, the chemical principles of this polymer-induced silicification remain unclear. Here, we used a biomimetic polycation-polyanion system to study polymer-induced silicification. We demonstrate that it is the polymer phase separation process, rather than silica-polymer interactions, which controls silica precipitation. Since ionic strength controls this electrostatic phase separation, it can be used to tune the morphology and structure of the precipitates. In situ cryo electron microscopy highlights the pivotal role of the hydrated polymer condensates in this process. These results pave the road for developing nanoscale morphologies of bioinspired silica based on the chemistry of liquid-liquid phase separation.

Influence of temperature and salt on coacervation in an aqueous mixture of poly-L-lysine (PLL) and poly-(sodium styrene sulfonate) (PSSNa).

The mixture of poly-L-lysine (PLL) and long-chain PSSNa can lead to the formation of soluble complexes depending on pH, PLL concentration, ionic strength, and temperature. The influence of these stimuli was studied by zetametry, dynamic and ultra-small-angle light scattering, and turbidimetric measurements. First of all, we studied the stoichiometry of complexation, and then considered the influence of salt concentration and temperature on the behavior of the mixture at different pH values. These findings have allowed us to conclude that the polyelectrolyte-polypeptide stoichiometry is controlled by electrostatic interactions between opposite charges. At mass ratios between 1.8 and 2.3 and with net charges close to neutrality, unstable complexes were formed and flocculated due to the hydrophobic attraction leading to macroscopic phase separation. The linear charge density of the complex is also controlled by the ionic strength. Higher CaCl2 concentrations reduce the complex stability and decrease the charge density, which leads to surface patch binding (SPB) at higher pH. Finally, the electrostatic interactions and strength of hydrogen bonds increased the stabilization of the complexes formed at temperatures lower than 45 °C. At temperatures higher than 45 °C, hydrophobic interactions became more dominant, causing a destabilization of the complexes.

The impact of pH and biopolymer ratio on the complex coacervation of Spirulina platensis protein concentrate with chitosan.

Spirulina platensis is one of the most significant multicellular blue-green Cyanobacterium microalgae with a high protein content. The complex coacervation as an encapsulation technique allows the formation of proteins with improved functional properties and thermal stability. In this study, the effects of pH and Spirulina platensis protein concentrate (SPPC)-chitosan ratio on complex coacervation formation were examined in terms of ζ-potential, turbidity, visual observation and microscopic images. Based on the results, the strongest interaction between SPPC and chitosan occurred at pH of 5.5 and SPPC-chitosan ratio of 7.5:1 with a precipitation in the test tubes. Stable dispersions were obtained at a pH range of 2-4 for the SPPC-chitosan ratio of 7.5:1 inhibiting the precipitation which occurs at individual SPPC solutions at this pH range. Characteristic organic groups in the individual SPPC and chitosan solutions as well as the SPPC-chitosan coacervate formed at the optimal conditions were identified by using Fourier Transform Infrared (FT-IR) spectroscopy technique. Furthermore, thermal stability of the individual SPPC and chitosan solutions and the SPPC-chitosan coacervates were investigated using differential scanning calorimetry (DSC). The glass transition temperature and enthalpy were 209.5 °C and - 3.414 W/g for the complex coacervates and 180.5 °C and - 0.877 W/g for SPPC. It means that complex coacervation provided more thermally-stable SPPC in chitosan-SPPC coacervate than that of the individual SPPC. Our results might have important implications for the utilization of Spirulina platensis proteins especially for acidic beverage applications.

Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us.

Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell-a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated "membraneless organelles" that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid-liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein-containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted.

Recent Advances in Astaxanthin Micro/Nanoencapsulation to Improve Its Stability and Functionality as a Food Ingredient.

Astaxanthin is a carotenoid produced by different organisms and microorganisms such as microalgae, bacteria, yeasts, protists, and plants, and it is also accumulated in aquatic animals such as fish and crustaceans. Astaxanthin and astaxanthin-containing lipid extracts obtained from these sources present an intense red color and a remarkable antioxidant activity, providing great potential to be employed as food ingredients with both technological and bioactive functions. However, their use is hindered by: their instability in the presence of high temperatures, acidic pH, oxygen or light; their low water solubility, bioaccessibility and bioavailability; their intense odor/flavor. The present paper reviews recent advances in the micro/nanoencapsulation of astaxanthin and astaxanthin-containing lipid extracts, developed to improve their stability, bioactivity and technological functionality for use as food ingredients. The use of diverse micro/nanoencapsulation techniques using wall materials of a different nature to improve water solubility and dispersibility in foods, masking undesirable odor and flavor, is firstly discussed, followed by a discussion of the importance of the encapsulation to retard astaxanthin release, protecting it from degradation in the gastrointestinal tract. The nanoencapsulation of astaxanthin to improve its bioaccessibility, bioavailability and bioactivity is further reviewed. Finally, the main limitations and future trends on the topic are discussed.