Protein Phase Behavior Research Articles

Extraction of mucilage polysaccharides from chia seed by hydrophobic deep eutectic solvents-based three-phase partitioning system: A phase behavior-driven approach

By incorporating the hydrophobic deep eutectic solvents (DESs) into the three-phase partitioning (TPP) technique, a TPP-based method was developed to extract the chia seed polysaccharide (CSP) from chia seed. Through a single-factor experiment and response-surface model, the optimal condition for the TPP extraction was determined as DES composed of dodecanoic acid and octanoic acid in a 1:1 M ratio, (NH4)2SO4 concentration of 32.86 %, crude extract–DES ratio of 0.93 (v/v), aqueous phase pH of 4.38, extraction temperature of 35 °C, and extraction time of 10 min. The polysaccharide yield of the constructed TPP method is 8.65 %, which is higher than the conventional water extraction method (yield is 6.96 %). Molecular dynamics simulations reveal the phase behavior of proteins and polysaccharides in the TPP system, showing that noncovalent interactions play a crucial role in the TPP system. The CSP obtained by the TPP method exhibits distinctive composition, structural, physicochemical, and functional properties, leading to improved thermal stability, rheological behavior, and antioxidant performance. Compared with the traditional extraction method, efficient extraction of CSP can be achieved flexibly using the proposed TPP approach, resulting in high yield and quality of CSP, which provides a new path for the large-scale utilization of chia seed.

The Effects of Electric Fields on Protein Phase Behavior and Protein Crystallization Kinetics.

We experimentally studied the effects of an externally applied electric field on protein crystallization and liquid-liquid phase separation (LLPS) and its crystallization kinetics. For a surprisingly weak alternating current (AC) electric field, crystallization was found to occur in a wider region of the phase diagram, while nucleation induction times were reduced, and crystal growth rates were enhanced. LLPS on the contrary was suppressed, which diminishes the tendency for a two-step crystallization scenario. The effect of the electric field is ascribed to a change in the protein-protein interaction potential.

Effective interactions in protein solutions with and without clustering

Protein interactions in solution and the question of cluster formation are of paramount importance to a wide range of research fields and industrial applications. Protein clustering can arise due to a delicate interplay between the short-range attraction and the long-range repulsion in the interaction potential. This study employs small angle X-ray scattering (SAXS) to examine the clustering behavior of three protein systems in solution: bovine serum albumin (BSA), bovine β-lactoglobulin (BLG) and lysozyme (LYZ) under varying protein concentrations, ionic strengths and temperatures. The goal is to elucidate the influence of the attractive potential’s nature on clustering behavior. BSA serves as a reference system, where electrostatic repulsions dominate the effective interactions, resulting in a mean protein–protein distance as a function of protein concentration which follows the 1/3 power law and precludes protein cluster formation. In contrast, BLG and LYZ solutions exhibit exponents less than 1/3, indicating concentration-dependent cluster formation. The effective interactions are well described using a two-Yukawa type potential comprising a long-ranged repulsion and a short-ranged attraction. While increasing ionic strength by adding NaCl reduced repulsion and correlation in all three systems, the response of protein solutions to ionic strength differed for clustering systems of BLG and LYZ. BLG solutions exhibited a reduced attraction with increasing ionic strength, leading to cluster dissolution, while LYZ solutions experienced enhanced attraction, favoring larger cluster formation. Notably, BLG solutions showed minimal temperature dependence, while LYZ solutions exhibited increased attraction with decreasing temperature, further promoting cluster formation. These results demonstrate the crucial role of short-ranged attractions of specific nature in determining protein clustering behavior. A thorough understanding of protein interactions is essential for predicting protein clustering and phase behavior.

Reversible disulfide bond crosslinks as tunable levers of phase separation in designer biomolecular condensates.

Biomolecular condensates (BCs) are membraneless hubs enriched in proteins and nucleic acids that have become important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is important in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the last decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to determine the involvement of disulfide crosslinks and their redox sensitivity in BCs, we designed a 'stickers and spacers' model of phase-separating peptides interspersed with cysteines. Through biophysical investigations, we learned that cysteines promote liquid-liquid phase separation in oxidizing conditions and perpetuate liquid condensates through disulfide crosslinks, which can be reversibly tuned with redox chemistry. By varying the composition of cysteines, subtle but distinct changes in the viscoelastic behavior of the condensates were observed. Empirically, we conclude that cysteines are neither stickers nor spacers but function as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the role of cysteines in protein phase behavior and the potential to develop tunable, redox-sensitive viscoelastic materials.

From adsorption to crystallization of proteins: Evidence for interface-assisted nucleation

Protein crystallization is among the key processes in biomolecular research, but the underlying mechanisms are still elusive. Here, we address the role of inevitable interfaces for the nucleation process. Quartz crystal microbalance with dissipation monitoring (QCM-D) with simultaneously optical microscopy, confocal microscopy, and grazing-incidence small angle X-rays scattering (GISAXS) were employed to investigate the temporal behavior from the initial stage of protein adsorption to crystallization. Here we studied the crystallization of the Human Serum Albumin (HSA), the most abundant blood protein, in the presence of a charged surface and a trivalent salt. We found evidence for interface-assisted nucleation of crystals. The kinetic stages involved are initial adsorption followed by enhanced adsorption after longer times, subsequent nucleation, and finally crystal growth. The results highlight the importance of interfaces for protein phase behavior and in particular for nucleation.

Liquid–liquid phase separation of the prion protein is regulated by the octarepeat domain independently of histidines and copper

Liquid-liquid phase separation (LLPS) of the mammalian prion protein is mainly driven by its intrinsically disordered N-terminal domain (N-PrP). However, the specific intermolecular interactions that promote LLPS remain largely unknown. Here, we used extensive mutagenesis and comparative analyses of evolutionarily distant PrP species to gain insight into the relationship between protein sequence and phase behavior. LLPS of mouse PrP is dependent on two polybasic motifs in N-PrP that are conserved in all tetrapods. A unique feature of mammalian N-PrP is the octarepeat domain with four histidines that mediate binding to copper ions. We now show that the octarepeat is critical for promoting LLPS and preventing formation of PrP aggregates. Amphibian N-PrP, which contains the polybasic motifs but lacks a repeat domain and histidines, does not undergo LLPS and forms non-dynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating phase transition of PrP. This activity of the octarepeat domain was neither dependent on the four highly conserved histidines nor on copper binding. Instead, the regularly spaced tryptophan residues were critical for regulating LLPS, presumably via cation-π interactions with the polybasic motifs. Our study reveals a novel role for the tryptophan residues in the octarepeat in controlling phase transition of PrP and indicates that the ability of mammalian PrP to undergo LLPS has evolved with the octarepeat in the intrinsically disordered domain, but independently of the histidines.

Effect of pH and biopolymer ratio on phase behavior, rheology, and structural characteristics of pea protein isolate‐locust bean gum coacervates

AbstractBackgroundInteraction between plant‐based proteins and polysaccharides depends on several factors. This work reports the influence of pH and biopolymer ratios on the protein (pea protein isolate, PPI)‐polysaccharide (locust bean gum, LBG) coacervates. Electrostatic interaction and hydrogen bond between the biopolymers strongly influenced the formation of the coacervates.ResultsThe optimum coacervate conditions were observed at pH 4.5 and PPI:LBG ratio of 5:1. The coacervates showed a honeycomb porous architecture having an amorphous nature. Complex coacervates showed a significant elevation of denaturation temperature as compared to biopolymers alone.ConclusionThese results indicates that PPI‐LBG coacervates can be used as an effective biomaterial for encapsulating heat‐sensitive bioactive compounds and other multiple uses in food processing.

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

Condensate interfaces can accelerate protein aggregation

Protein aggregates, formed from the assembly of aberrant, misfolded proteins, are a hallmark of neurodegenerative diseases. Disease-associated aggregates such as mutant Huntingtin polyQ inclusions, are typically enriched in p62/SQSTM1, an oligomeric protein that binds to and sequesters aberrant proteins. p62 has been suggested to sequester proteins through formation of liquid-like biomolecular condensates, but the physical mechanisms by which p62 condensates may regulate pathological protein aggregation remain unclear. Here, we use a light-inducible biomimetic condensate system to show that p62 condensates enhance coarsening of mutant polyQ aggregates through interface-mediated sequestration, which accelerates polyQ accumulation into larger aggregates. However, the resulting large aggregates accumulate polyubiquitinated proteins, which depletes free p62, ultimately suppressing further p62 condensation. This dynamic interplay between interface-mediated coarsening of solid aggregates and downstream consequences on the phase behavior of associated regulatory proteins could contribute to the onset and progression of protein aggregation diseases.

Probing ion-binding at a protein interface: Modulation of protein properties by ionic liquids

Ions are important to modulate protein properties, including solubility and stability, through specific ion effects. Ionic liquids (ILs) are designer salts with versatile ion combinations with great potential to control protein properties. Although protein-ion binding of common metals is well-known, the IL effect on proteins is not well understood. Here, we employ the model protein lysozyme in dilute and concentrated IL solutions to determine the specific ion binding effect on protein phase behaviour, activity, size and conformational change, aggregation and intermolecular interactions. A combination of spectroscopic techniques, activity assays, small-angle X-ray scattering, and crystallography highlights that ILs, particularly their anions, bind to specific sites in the protein hydration layer via polar contacts on charged, polar and aromatic residues. The specific ion binding can induce more flexible loop regions in lysozyme, while the ion binding in the bulk phase can be more dynamic in solution. Overall, the protein behaviour in ILs depends on the net effect of nonspecific interactions and specific ion binding. Compared to formate, the nitrate anion induced high protein solubility, low activity, elongated shape and aggregation, which is largely owing to its higher propensity for ion binding. These findings provide new insights into protein-IL binding interactions and using ILs to modulate protein properties.

Probing pressure-driven protein phase behavior via in situ high-pressure scattering methods

Probing pressure-driven protein phase behavior via <i>in situ</i> high-pressure scattering methods

Simple Coacervation of Guanidinium-Containing Polymers Induced by Monovalent Salt

Liquid–liquid phase separation (LLPS) of macromolecules, called coacervation, is induced by non-covalent intermolecular associations in aqueous solutions, which is frequently observed in biological processes including cellular compartmentation, signaling, and regulation. Recently, associative interactions between π-conjugated residues have emerged to induce LLPS, along with electrostatic interaction. An arginine residue having a positively charged guanidinium group plays a pivotal role in protein phase behavior because the orientational amphiphilicity of guanidinium groups allows face-to-face π–π stacking despite Coulombic repulsion. In this study, we investigate the simple coacervates of guanidinium-functionalized polyelectrolytes (i.e., poly-arginine) in aqueous media as a function of salt concentration, salt type, and temperature to understand the exclusive role of π-conjugated moieties. Contrary to ammonium-functionalized polyelectrolyte (i.e., poly-lysine) solutions, guanidinium-functionalized polyelectrolyte solutions become turbid by adding monovalent salts and exhibit the upper critical solution temperature (UCST) behavior; the critical temperature is harnessed by salt concentration and salt type. Although a π–π stacked guanidinium pair can exist at a lower salt concentration, LLPS occurs when the number of intermolecular guanidinium pairs goes beyond a critical value to produce the network structure. Furthermore, the enthalpically favored π–π interaction directly affects the bulk rheological behavior and the interfacial property of the coacervates. Our findings provide insights into the underlying interactions of protein phase separation and shed new light on the critical role of π–π stacking in the biological process and material design.

Insights into the solubility of D-crystallin from multiscale atomistic simulations.

The molecular basis underlying the rich phase behavior of globular proteins remains poorly understood. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics and interprotein interactions of D-crystallin and its P23T-R36S mutant, which drastically limits the protein solubility, at both infinite dilution and at a concentration where the mutant fluid phase and crystalline phase coexist. We find that while the mutant conserves the protein fold, changes to the surface exposure of residues in the neighborhood of residue-36 enhance protein-protein interactions and develop specific protein-protein contacts found in the protein crystal lattice.

WNK1, a molecular crowding sensor, links phase separation to cellular physiological stress.

WNK1, a molecular crowding sensor, links phase separation to cellular physiological stress.

Comparison of Biomolecular Condensate Localization and Protein Phase Separation Predictors

Research in the field of biochemistry and cellular biology has entered a new phase due to the discovery of phase separation driving the formation of biomolecular condensates, or membraneless organelles, in cells. The implications of this novel principle of cellular organization are vast and can be applied at multiple scales, spawning exciting research questions in numerous directions. Of fundamental importance are the molecular mechanisms that underly biomolecular condensate formation within cells and whether insights gained into these mechanisms provide a gateway for accurate predictions of protein phase behavior. Within the last six years, a significant number of predictors for protein phase separation and condensate localization have emerged. Herein, we compare a collection of state-of-the-art predictors on different tasks related to protein phase behavior. We show that the tested methods achieve high AUCs in the identification of biomolecular condensate drivers and scaffolds, as well as in the identification of proteins able to phase separate in vitro. However, our benchmark tests reveal that their performance is poorer when used to predict protein segments that are involved in phase separation or to classify amino acid substitutions as phase-separation-promoting or -inhibiting mutations. Our results suggest that the phenomenological approach used by most predictors is insufficient to fully grasp the complexity of the phenomenon within biological contexts and make reliable predictions related to protein phase behavior at the residue level.

Examining compositional variability of giant unilamellar vesicles via secondary ion mass spectrometry.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. However, there may be significant compositional variability between individual GUVs, either due to lipid sorting during film hydration or from vesicle budding following GUV formation. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary POPC/DSPC/CHOL mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in a single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H−/13C−, which is highly sensitive to compositional changes between individual GUVs and circumvents the need for calibration using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display compositional variability on the order of several mole percent.

Multi-scale molecular simulation of random peptide phase separation and its extended-to-compact structure transition driven by hydrophobic interactions.

Intrinsically disordered proteins (IDPs) often undergo liquid-liquid phase separation (LLPS) and form membraneless organelles or protein condensates. One of the core problems is how do electrostatic repulsion and hydrophobic interactions in peptides regulate the phase separation process? To answer this question, this study uses random peptides composed of positively charged arginine (Arg, R) and hydrophobic isoleucine (Ile, I) as the model systems, and conduct large-scale simulations using all atom and coarse-grained model multi-scale simulation methods. In this article, we investigate the phase separation of different sequences using a coarse-grained model. It is found that the stronger the electrostatic repulsion in the system, the more extended the single-chain structure, and the more likely the system forms a low-density homogeneous phase. In contrast, the stronger the hydrophobic effect of the system, the more compact the single-chain structure, the easier phase separation, and the higher the critical temperature of phase separation. Overall, by taking the random polypeptides composed of two types of amino acid residues as model systems, this study discusses the relationship between the protein sequence and phase behaviour, and provides theoretical insights into the interactions within or between proteins. It is expected to provide essential physical information for the sequence design of functional IDPs, as well as data to support the diagnosis and treatment of the LLPS-associated diseases.

Liquid-Liquid Phase Separation Prediction of Proteins in Salt Solution by Deep Neural Network.

Liquid-liquid phase separation (LLPS) underlies the formation of membrane-free organelles in eukaryotic cells and plays an important role in the development of some diseases. The phase boundary of metastable liquid-liquid phase separation as well as the cloud point temperature of some globular proteins characterize the phase behavior of proteins and have been widely studied theoretically and experimentally. In the present study, we used a regression and classification neural network to deal with the phase behavior of lysozyme and bovine serum albumin (BSA). We predicted the cloud point temperature and solubility of a lysozyme solution containing sodium chloride by regression and the reentrant phase behavior of BSA in YCl3 solution containing a surfactant dodecyl dimethyl amine oxide (DDAO) by classification. Specifically, our network model is capable of predicting (a) the solubility of lysozyme in the range: pH 4.0-5.4, temperature 0-25 °C, and NaCl concentration 2-7% (w/v); (b) the cloud point temperature of lysozyme in the range: pH 4.0-4.8, NaCl concentration 2-7%, and lysozyme concentration 0-400 mg/mL; and (c) the phase behavior of BSA in the range: DDAO 1-60 mM, BSA 30-100 mg/mL, and YCl3 1-20 mM. We experimentally tested the model at some prediction points with a high accuracy, which means that deep neural networks can be applicable in qualitative and quantitive analysis of liquid-liquid phase separation.

Interaction and phase behavior of whey protein and propylene glycol alginate complex condensates

Whey protein (WP) is ubiquitously applied in food products, but its sensitivity to food processing conditions has limited its application. Herein, we chose propylene glycol alginate (PGA) to combine with WP to enhance its stability. The ideal ratio of WP/PGA for coacervation was 3:1, and the soluble complex and insoluble complex were formed at pH 5.2 (pHc) and pH 4.4 (pHφ1) at this ratio, respectively. The UV absorption spectra, fluorescence spectra, and XRD results revealed that the interaction between PGA and WP changed the tertiary conformation of WP. The FTIR and molecular docking results suggested electrostatic interactions, hydrogen bonding and hydrophobic interactions were all involved in the formation of WP-PGA complexes, and the thermal stability of WP was improved based on the DSC results. These findings supported PGA to keep dairy products stable and transparent at the isoelectric point and WP-PGA complexes could be applied in encapsulating bioactive substances.

Simplified choice of suitable excipients within biologics formulation design using protein-protein interaction- and water activity-maps

Still today, high-concentration protein formulations are often developed based on high-throughput experimental screening approaches. Although likely delivering working formulations, these approaches do not lead to a deep/mechanistic understanding of the protein phase behavior in solution. Within this work, we thus optimized and enhanced a recent approach for an initial low effort selection of potential excipients and excipient mixtures to be used in high-concentration protein formulations. This approach considers both: molecular interactions and thermodynamic determinants to access the phase behavior of the proteins in solution, as well as pharmaceutical engineering boundaries (such as osmotic pressure and osmolality) to deliver on optimal formulation conditions. Water activity coefficient γW-calculations (used to describe the protein environment in solution), unfolding temperature (conformational stability) and protein-protein interactions (colloidal stability) are used as determinants. Amino acids (20 proteinogenic amino acids), selected amino acid mixtures, as well as mixtures of amino acids and trehalose (l-arginine-trehalose; l-histidine-trehalose) are considered as model excipients. The approach is extends by studying the long-term stability of the predicted formulation conditions for a γ-globulin from human blood and denosumab. The results reveal, that by combining protein-specific experiments as well as model-based studies for the selection of excipient mixtures in high concentration protein formulations, the effort as well as the resource requirements can be reduced significantly.