Reentrant Condensation Research Articles

Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes.

In eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a windowlike phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using ribonucleoprotein-inspired polypeptides with low-complexity RNA-binding sequences as well as an archetypal disordered RNP, fused in sarcoma, as model systems, we investigate the molecular driving forces underlying this nonmonotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC behavior of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, control the two-phase coexistence regime, regulate material properties of polypeptide-RNA condensates, and oppose condensate reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloidlike cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

Specific Ion Effects of Trivalent Cations on the Structure and Charging State of β-Lactoglobulin Adsorption Layers.

The properties of proteins at interfaces are important to many processes as well as in soft matter materials such as aqueous foam. Particularly, the protein interfacial behavior is strongly linked to different factors like the solution pH or the presence of electrolytes. Here, the nature of the electrolyte ions can significantly modify the interfacial properties of proteins. Therefore, molecular level studies on interfacial structures and charging states are needed. In this work, we addressed the effects of Y3+ and Nd3+ cations on the adsorption of the whey protein β-lactoglobulin (BLG) at air-water interfaces as the function of electrolyte concentration. Both cations caused very similar but dramatic changes at the interface and in the bulk solution. Here, measurements of the electrophoretic mobility and with vibrational sum-frequency generation (SFG) spectroscopy were applied and consistently showed a reversal of the BLG net charge at remarkably low ion concentrations of 30 (bulk) and 40 (interface) μM of Y3+ or Nd3+ for a BLG concentration of 15 μM. SFG spectra of carboxylate stretching vibrations from Asp or Glu residues of interfacial BLG showed significant changes in the resonance frequency, which we associate to specific and efficient binding of Y3+ or Nd3+ ions to the proteins carboxylate groups. Characteristic reentrant condensation for BLG moieties with bound trivalent ions was found in a broad concentration range around the point of zero net charge. The highest colloidal stability of BLG was found for ion concentrations <20 μM and >50 μM. Investigations on macroscopic foams from BLG solutions revealed the existence of structure-property relations between the interfacial charging state and the foam stability. In fact, a minimum in foam stability at 20 μM ion concentration was found when the interfacial net charge was negligible. At this concentration, we propose that the persistent BLG molecules and weakly charged BLG aggregates drive foam stability, while outside the bulk reentrant zone the electrostatic disjoining pressure inside foam lamellae dominates foam stability. Our results provide new information on the charge reversal at the liquid-gas interface of protein/ion dispersions. Therefore, we see our findings as an important step in the clarification of reentrant condensation effects at interfaces and their relevance to foam stability.

Interaction of Anionic Bulk Nanobubbles with Cationic Liposomes: Evidence for Reentrant Condensation.

We investigated the interaction of bulk nanobubbles with cationic liposomes composed of 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine and anionic liposomes assembled from 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol). We employed dynamic light scattering and fluorescence microscopy to investigate both the hydrodynamic and electrophoretic properties of the nanobubble/liposome complexes. These optical techniques permit direct visualization of structural changes as a function of the bubble/liposome ratio. We observed reentrant condensation with cationic liposomes and gas nucleation with anionic liposomes. This is the first report of charge inversion and reentrant condensation of cationic liposomes induced by bulk nanobubbles.

Controlling Phase Separation of Lysozyme with Polyvalent Anions.

The ability of polyvalent anions to influence protein-protein interactions and protein net charge was investigated through solubility and turbidity experiments, determination of osmotic second virial coefficients ( B22), and ζ-potential values for lysozyme solutions. B22 values showed that all anions reduce protein-protein repulsion between positively charged lysozyme molecules, and those anions with higher net valencies are more effective. The polyvalent anions pyrophosphate and tripolyphosphate were observed to induce protein reentrant condensation, which has been previously observed with negatively charged proteins in the presence of trivalent cations. Reentrant condensation is a phenomenon in which low concentrations of polyvalent ions induce protein precipitation, but further increasing polyvalent ion concentration causes the protein precipitate to resolubilize. Interestingly, citrate does not induce lysozyme reentrant condensation despite having a similar charge, size, and shape to pyrophosphate. We observe qualitative differences in protein behavior when compared against negatively charged proteins in solutions of trivalent cations. The polyphosphate ions induce a much stronger protein-protein attraction, which correlates with the occurrence of a liquid-gel transition that replaces the liquid-liquid transition observed with trivalent cations. The results indicate that solutions of polyphosphate ions provide a model system for exploring the link between the protein-phase diagram and model interaction potentials and also highlight the importance that ion-specific effects can have on protein solubility.

Optical responses of BSA protein under re-entrant condensation in presence of trivalent ions

Fluorescence behavior of globular protein like BSA is studied under the re-entrant condensation in the presence of trivalent salts like YCl3 and LaCl3. BSA shows a re-entrant behaviour in solution where a specific turbid phase is formed between the two critical concentrations of the trivalent salts. Optical behaviors of proteins are explored by UV–Vis, photoluminescence and time resolved photoluminescence (TRPL) spectroscopy, whereas hydrodynamic size is obtained from the dynamic light scattering (DLS) technique. BSA shows maximum emission intensity at ≈338 nm for an excitation at 278 nm, however, the value of the peak intensity of emission gradually increases with the increase in salt concentration and becomes maximum at the turbid phase but for higher salt concentration as the re-dissolution takes place, the emission intensity again decreases. TRPL study suggests that the decay time of BSA under re-entrant condensation is correlated with the salt concentration, whereas DLS study shows that the hydrodynamic size gradually increases up to a certain salt concentration where turbid phase is formed and then again decreases for higher salt concentrations. The variation of emission intensity of BSA nearly follows the modification of diffusion coefficient or hydrodynamic size and the phase transition behaviors.

Nanoparticle–nanobubble interactions: Charge inversion and re-entrant condensation of amidine latex nanoparticles driven by bulk nanobubbles

HypothesisThe stability of colloidal suspensions can be influenced by supersaturation of the supporting electrolyte with gas. It has been proposed that this effect can be attributed to the formation of nanobubbles on the surface of the colloidal particles, in turn influencing DLVO forces. While previous interpretations have focused primarily on van der Waals interactions, probing positively charged particles can provide complementary insight into electrostatic interactions. ExperimentsHigh-power water electrolysis creates an aqueous solution supersaturated with oxygen and hydrogen. We study the ability of this solution to influence the electrophoretic properties of positive nanoparticles as a function of the particle-gas ratio. Both the ζ-potential and the effective hydrodynamic diameter of the resulting nanoentities were studied using dynamic light scattering for a range of nanoparticle sizes. FindingsGas-saturated solution interacts strongly with positive nanoparticles by decreasing and ultimately reversing the sign of their ζ-potential, which we attribute to the nucleation of negatively charged bubbles at the solid-liquid interface. This leads to re-entrant condensation of the particles near their point of zero charge, as directly observed via an increase in hydrodynamic diameter and macroscopic aggregation. These results indicate that modulation of electrostatic interactions can be the dominant mechanism for gas-particle interactions in these systems.

Reentrant Phase Behavior in Protein Solutions Induced by Multivalent Salts: Strong Effect of Anions Cl- Versus NO3.

In this work, the effects of the two anions Cl- and NO3- on the phase behavior of bovine serum albumin (BSA) in solution with trivalent salts are compared systematically. In the presence of trivalent metal salts, negatively charged proteins such as BSA in solution undergo a reentrant condensation (RC) phase behavior, which has been established for several proteins with chlorides of trivalent salts. Here, we show that replacing Cl- by NO3- leads to a marked change in the phase behavior. The effect is investigated for the two different cations Y3+ and La3+. The salts are thus YCl3, Y(NO3)3, LaCl3, and La(NO3)3. The experimental phase behavior shows that while the chloride salts induce both liquid-liquid phase separation (LLPS) and RC, the nitrate salts also induce LLPS, but RC becomes partial with La(NO3)3 and disappears with Y(NO3)3. The observed phase behavior is rationalized by effective protein-protein interactions which arecharacterized using small-angle X-ray scattering. The results based on the reduced second virial coefficients B2/ B2HS and 1/ I( q → 0) demonstrate that the NO3- salts induce a stronger attraction than the Cl- salts. Overall, the effective attraction, the width of the condensed regime in the RC phase diagram, and the nature of LLPS follow the order LaCl3 < YCl3 < La(NO3)3 < Y(NO3)3. Despite the decisive role of cations in RC phase behavior, isothermal titration calorimetry measurements indicate that replacing anions does not significantly influence the cation binding to proteins. The experimental results observed are discussed based on an "enhanced Hofmeister effect" including electrostatic and hydrophobic interactions between protein-cation complexes.

Effective potential between negatively charged patches on acidic proteins immersed in various electrolyte solutions.

Effective interactions between O-sized anions in various electrolyte solutions were calculated by using the integral equation theory with some simple models. The results indicated that only multivalent cations mediated a strong effective attraction between O-sized anions at a certain concentration. The effective interaction turned from repulsive to attractive as the electrolyte concentration increased, and the effective attraction decreased when more electrolyte was added. Moreover, the effective interactions between O-sized anions in the electrolyte solution did not present a long repulsive tail, although the effective attraction caused by the divalent cations appeared. By contrast, the effective attraction mediated by monovalent cations and the reentrant behavior did not appear and the effective interaction was basically repulsive. These behaviors agree with the experimental results for reentrant condensation of acidic proteins in various electrolyte solutions. The calculated results suggest that the dissociated carboxylic acidic groups on the proteins form attractive patches between proteins under certain concentration conditions.

Overcharging and reentrant condensation of thermoresponsive ionic microgels.

We investigated the complexation of thermoresponsive anionic poly(N-isopropylacrylamide) (PNiPAM) microgels and cationic ε-polylysine (ε-PLL) chains. By combining electrophoresis, light scattering, transmission electron microscopy (TEM) and dielectric spectroscopy (DS) we studied the adsorption of ε-PLL onto microgel networks and its effect on the stability of suspensions. We show that the volume phase transition (VPT) of microgels triggers a large polyion adsorption. Two interesting phenomena with unique features occur: a temperature-dependent microgel overcharging and a complex reentrant condensation. The latter may occur at fixed polyion concentration, when temperature is raised above the VPT of microgels, or by increasing the number density of polycations at fixed temperature. TEM and DS measurements unambiguously show that short PLL chains adsorb onto microgels and act as electrostatic glue above the VPT. By performing thermal cycles, we further show that polyion-induced clustering is a quasi-reversible process: within the time of our experiments large clusters form above the VPT and partially re-dissolve as the mixtures are cooled down. Finally we give a proof that the observed phenomenology is purely electrostatic in nature: an increase of the ionic strength gives rise to polyion desorption from the microgel outer shell.

Multivalent-Ion-Activated Protein Adsorption Reflecting Bulk Reentrant Behavior.

Protein adsorption at the solid-liquid interface is an important phenomenon that often can be observed as a first step in biological processes. Despite its inherent importance, still relatively little is known about the underlying microscopic mechanisms. Here, using multivalent ions, we demonstrate the control of the interactions and the corresponding adsorption of net-negatively charged proteins (bovine serum albumin) at a solid-liquid interface. This is demonstrated by ellipsometry and corroborated by neutron reflectivity and quartz-crystal microbalance experiments. We show that the reentrant condensation observed within the rich bulk phase behavior of the system featuring a nonmonotonic dependence of the second virial coefficient on salt concentration c_{s} is reflected in an intriguing way in the protein adsorption d(c_{s}) at the interface. Our findings are successfully described and understood by a model of ion-activated patchy interactions within the framework of the classical density functional theory. In addition to the general challenge of connecting bulk and interface behavior, our work has implications for, inter alia, nucleation at interfaces.

Anomalous Protein-Protein Interactions in Multivalent Salt Solution.

The stability of aqueous protein solutions is strongly affected by multivalent ions, which induce ion-ion correlations beyond the scope of classical mean-field theory. Using all-atom molecular dynamics (MD) and coarse grained Monte Carlo (MC) simulations, we investigate the interaction between a pair of protein molecules in 3:1 electrolyte solution. In agreement with available experimental findings of "reentrant protein condensation", we observe an anomalous trend in the protein-protein potential of mean force with increasing electrolyte concentration in the order: (i) double-layer repulsion, (ii) ion-ion correlation attraction, (iii) overcharge repulsion, and in excess of 1:1 salt, (iv) non Coulombic attraction. To efficiently sample configurational space we explore hybrid continuum solvent models, applicable to many-protein systems, where weakly coupled ions are treated implicitly, while strongly coupled ones are treated explicitly. Good agreement is found with the primitive model of electrolytes, as well as with atomic models of protein and solvent.

Reentrant condensation transition in a two species driven diffusive system

We study an interacting box-particle system on a one-dimensional periodic ring involving two species of particles A and B. In this model, from a randomly chosen site, a particle of species A can hop to its right neighbor with a rate that depends on the number of particles of the species B at that site. On the other hand, particles of species B can be transferred between two neighboring sites with rates that depends on the number of particles of species B at the two adjacent sites–this process however can occur only when the two sites are devoid of particles of the species A. We study condensation transition for a specific choice of rates and find that the system shows a reentrant phase transition of species A –the species A passes successively through fluid–condensate–fluid phases as the coupling parameter between the dynamics of the two species is varied. On the other hand, the transition of species B is from condensate to fluid phase and hence does not show reentrant feature.

Strong Isotope Effects on Effective Interactions and Phase Behavior in Protein Solutions in the Presence of Multivalent Ions.

In this article, we have studied the influence of the isotopic composition of the solvent (H2O or D2O) on the effective interactions and the phase behavior of the globular protein bovine serum albumin in solution with two trivalent salts (LaCl3 and YCl3). Protein solutions with both salts exhibit a reentrant condensation phase behavior. The condensed regime (regime II) in between two salt concentration boundaries (c* < cs < c**) is significantly broadened by replacing H2O with D2O. Within regime II, liquid-liquid phase separation (LLPS) occurs. The samples that undergo LLPS have a lower critical solution temperature (LCST). The value of LCST decreases significantly with increasing solvent fraction of D2O. The effective protein-protein interactions characterized by small-angle X-ray scattering demonstrate that although changing the solvent has negligible effects below c*, where the interactions are dominated by electrostatic repulsion, an enhanced effective attraction is observed in D2O above c*, consistent with the phase behavior observed. As the LCST-LLPS is an entropy-driven phase transition, the results of this study emphasize the role of entropy in solvent isotope effects.

Salt-induced reentrant stability of polyion-decorated particles with tunable surface charge density

The electrostatic complexation between DOTAP/DOPC unilamellar liposomes and an oppositely charged polyelectrolyte (NaPA) has been investigated in a wide range of the liposome surface charge density. We systematically characterized the reentrant condensation and the charge inversion of polyelectrolyte-decorated liposomes by means of dynamic light scattering and electrophoresis. We explored the stability of this model polyelectrolyte/colloid system at different values of the surface charge of the bare liposomes and by changing two independent control parameters of the suspensions: the polyelectrolyte/colloid charge ratio and the ionic strength of the aqueous suspending medium. The progressive addition of neutral DOPC lipid within the liposome membrane gave rise to an interesting phenomenon which has not been observed previously: the stability diagram of the suspensions showed a novel reentrance due to the crossing of the desorption threshold of the polyelectrolyte. Indeed, at fixed charge density of the bare DOTAP/DOPC liposomes and for a wide range of polyion concentrations, we showed that the simple electrolyte addition first (low salt regime) destabilizes the suspensions because of the enhanced screening of the residual repulsion between the complexes, and then (high salt regime) determines the onset of a new stable phase, originated by the absence of polyelectrolyte adsorption on the particle surfaces. We show that the observed phenomenology can be rationalized within the modified Velegol–Thwar model for heterogeneously charged particles and that the polyelectrolyte desorption fits well the predictions of the adsorption theory of Winkler and Cherstvy [1]. Our findings unambiguously support the picture of the reentrant condensation as driven by the correlated adsorption of the polyelectrolyte chains on the particle surface, providing interesting insights into possible mechanisms for tailoring complex colloids via salt-induced effects.

Polyethylene glycol and divalent salt-induced DNA reentrant condensation revealed by single molecule measurements

In this study, we investigated the DNA condensation induced by polyethylene glycol (PEG) with different molecular weights (PEG 600 and PEG 6000) in the presence of NaCl or MgCl2 by using magnetic tweezers (MT) and atomic force microscopy (AFM). The MT measurements show that with increasing NaCl concentration, the critical condensation force in the PEG 600-DNA or PEG 6000-DNA system increased approximately linearly. PEG 6000 solution has a larger critical force than PEG 600 solution at a given NaCl concentration. In comparison, a parabolic trend of the critical condensation force was observed with increasing MgCl2 concentration, indicating that DNA undergoes a reentrant condensation. The AFM results show that the morphologies of the compacted DNA-PEG complexes depended on the salt concentration and were consistent with the MT results.

Ion-activated attractive patches as a mechanism for controlled protein interactions.

The understanding of protein interactions to control phase and nucleation behavior of protein solutions is an important challenge for soft matter, biological and medical research. Here, we present ion bridges of multivalent cations between proteins as an ion-activated mechanism for patchy interaction that is directly supported by experimental findings in protein crystals. A deep understanding of experimentally observed phenomena in protein solutions—including charge reversal, reentrant condensation, metastable liquid-liquid phase separation, cluster formation and different pathways of crystallization—is gained by an analytic model that directly displays parameter dependencies and physical connections. The direct connection between experiment and theory provides a conceptual framework for future experimental, computational and theoretical research on topics such as rational design of phase behavior and crystallization pathways on the basis of the statistical physics of patchy particles.

Nonclassical Pathways of Protein Crystallization in the Presence of Multivalent Metal Ions

Using bovine β-lactoglobulin as a model system, we have studied the crystallization pathways in the presence of the di- and trivalent salts ZnCl2 and YCl3. Previous work has shown that protein solutions undergo a reentrant condensation (RC) phase behavior in the presence of YCl3, i.e., a condensed phase occurs in between two boundary salt concentrations, c*< c**. In the presence of ZnCl2, c* also exists, but protein solutions with high salt concentrations do not become completely clear at higher protein concentrations (>20 mg/mL). We thus denote this diffuse transition as pseudo-c**. Small angle X-ray scattering measurements show that the effective interactions change from repulsion to attraction near c*, and attractive interactions dominate around pseudo–c**. Solutions near c* and pseudo-c** provide the optimized conditions for growth of high quality protein single crystals but with different pathways. While crystal growth near c* follows the classical one-step nucleation pathway, crystallization around pseudo-c** (for zinc) or c** (for yttrium) follows a nonclassical process with an intermediate phase appearing before crystallization. Furthermore, the intermediate phases strongly depend on the crystallization temperature. Samples with high salt concentrations exhibit a typical transition temperature, Ttr, below which the solutions become turbid. When crystallizing near Ttr, the intermediate phase consists of protein clusters; below Ttr, the intermediate phase corresponds to macroscopic protein aggregates which can further relax into a dense liquid phase before crystallization. However, the experimental data cannot distinguish whether nucleation occurs within or outside of the intermediate phase. Possible scenarios are discussed based on the equilibrium phase behavior of colloidal systems with a short-range attraction. The crystallographic analysis of the resulting crystals shows that metal ions are an integral part of the crystal lattice. Both types of metal ions can create new protein contacts in the crystal lattice via bridging; however, yttrium creates more bridging contacts compared to zinc.

Competing salt effects on phase behavior of protein solutions: tailoring of protein interaction by the binding of multivalent ions and charge screening.

The phase behavior of protein solutions is affected by additives such as crowder molecules or salts. In particular, upon addition of multivalent counterions, a reentrant condensation can occur; i.e., protein solutions are stable for low and high multivalent ion concentrations but aggregating at intermediate salt concentrations. The addition of monovalent ions shifts the phase boundaries to higher multivalent ion concentrations. This effect is found to be reflected in the protein interactions, as accessed via small-angle X-ray scattering. Two simulation schemes (a Monte Carlo sampling of the counterion binding configurations using the detailed protein structure and an analytical coarse-grained binding model) reproduce the shifts of the experimental phase boundaries. The results support a consistent picture of the protein interactions responsible for the phase behavior. The repulsive Coulomb interaction is varied by the binding of multivalent counterions and additionally screened by any increase of the ionic strength. The attractive interaction is induced by the binding of multivalent ions, most likely due to ion bridging between protein molecules. The overall picture of these competing interactions provides interesting insight into possible mechanisms for tailoring interactions in solutions via salt effects.

Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride.

Recent studies have outlined the use of eutectic solutions of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without solvent crystallization. The eutectic point in a (H2O)R(LiCl) system corresponds to R ≈ 7.3, and it is of interest to investigate whether less-concentrated aqueous solutions of LiCl could be used in low-temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low-temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins.

Reentrant condensation, liquid–liquid phase separation and crystallization in protein solutions induced by multivalent metal ions

Abstract We briefly summarize the recent progress in tuning protein interactions as well as phase behavior in protein solutions using multivalent metal ions. We focus on the influence of control parameters and the mechanism of reentrant condensation, the metastable liquid–liquid phase separation and classical vs. non-classical pathways of protein crystallization.