RESUMEN
Protein aggregation is a major hurdle in developing biopharmaceuticals, in particular protein formulation area, but plays a pivotal role in food products. Co-solvents are used to suppress protein aggregation in pharmaceutical proteins. On the contrary, aggregation is encouraged in the process of food product making. Thus, it is expected that co-solvents play a contrasting role in biopharmaceutical formulation and food products. Here, we show several examples that utilize co-solvents, e.g., salting-out salts, sugars, polyols and divalent cations in promoting protein-protein interactions. The mechanisms of co-solvent effects on protein aggregation and solubility have been studied on aqueous protein solution and applied to develop pharmaceutical formulation based on the acquired scientific knowledge. On the contrary, co-solvents have been used in food industries based on empirical basis. Here, we will review the mechanisms of co-solvent effects on protein-protein interactions that can be applied to both pharmaceutical and food industries and hope to convey knowledge acquired through research on co-solvent interactions in aqueous protein solution and formulation to those involved in food science and provide those involved in protein solution research with the observations on aggregation behavior of food proteins.
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Proteínas , Solventes , Proteínas/química , Proteínas/metabolismo , Solventes/química , Solubilidad , Agregado de Proteínas , HumanosRESUMEN
Reentrant condensation (RC) is a protein behavior in which the protein solution shifts between the one- and two-phase state more than twice by increasing a single parameter. Although RC would be a candidate mechanism for the physicochemical design of food additives, no realistic model has been established under diverse contaminants like food materials. Here, we found that a mixture of cola and milk yielded RC. At pH 3.2-3.6, cola induced milk condensation at 30-40%, while lower or higher concentrations of cola did not. Furthermore, we reduced this cola/milk system to two pure components, casein in milk and polyphosphate (polyP) in cola, and investigated the characteristics of casein concentration and zeta potential. This was the first experimental demonstration of RC occurrence in a multicomponent system. The well-characterized cola/milk system would explore both the universal nature of proteins and the industrial application of RC.
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Liquid-liquid phase separation (LLPS) and droplet formation by LLPS are key concepts used to explain compartmentalization in living cells. Protein contact to a membrane surface is considered an important process for protein organization in a liquid phase or during transition to a solid or liquid dispersion state. The direct experimental comprehensive investigation is; however, not performed on the surface-droplet interaction and phase transition. In the present study, we constructed simple and reproducible experiments to analyze the structural transition of aggregates and droplets in an ovalbumin (OVA) and lysozyme (LYZ) complex on glass slides with various coatings. The difference in droplet-surface interaction may only be important in the boundary region between aggregates and droplets of a protein mixture, as shown in the phase diagram. Co-aggregates of OVA-LYZ changed to droplet-like circular forms during incubation. In contrast, free l-lysine resulted in the uniform droplet-to-solid phase separation at lower concentrations and dissolved any structures at higher concentrations. These results represent the first phase-diagram-based analysis of the phase transition of droplets in a protein mixture and a comparison of surface-surface and small molecular-droplet structure interactions.
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Separación de Fases , Proteínas , Proteínas/químicaRESUMEN
Unstructured biological macromolecules have attracted attention as protein aggregation inhibitors in living cells. Some are characterized by their free structural configuration, highly charged, and water-soluble. However, the importance of these properties in inhibiting protein aggregation remains unclear. In this study, we investigated the effect of charged poly (amino acids), which mimic these properties, on aggregation of l-lactate dehydrogenase (LDH) and compared their effects to monomeric amino acids and folded proteins. LDH was stable and active at a neutral pH (~7) but formed inactive aggregates at acidic pH (< 6). Adding cationic polyelectrolytes of poly-l-lysine and poly-l-arginine suppressed the acid-induced aggregation and inactivation of LDH under acidic pH values. Adding monomeric amino acids and cationic folded proteins also prevented LDH aggregation but with lower efficacy than cationic polyelectrolytes. These results indicate that unstructured polyelectrolytes effectively stabilize unstable enzymes because they interact flexibly and multivalently with them. Our findings provide a simple method for stabilizing enzymes under unstable conditions.
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L-Lactato Deshidrogenasa , Agregado de Proteínas , Polielectrolitos/química , L-Lactato Deshidrogenasa/metabolismo , Proteínas , Aminoácidos/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) is essential to understanding the biomacromolecule compartmentalization in living cells and to developing soft-matter structures for chemical reactions and drug delivery systems. However, the importance of detailed experimental phase diagrams of modern LLPS systems tends to be overlooked in recent times. Even for the poly(l-lysine) (PLL)/ATP system, which is one of the most widely used LLPS models, any detailed phase diagram of LLPS has not been reported. Herein, we report the first phase diagram of the PLL/ATP system and demonstrate the feasibility of phase-diagram-based research design for understanding the physical properties of LLPS systems and realizing biophysical and medical applications. We established an experimentally handy model for the droplet formation-disappearance process by generating a concentration gradient in a chamber for extracting a suitable condition on the phase diagram, including the two-phase droplet region. As a proof of concept of pharmaceutical application, we added a human immunoglobulin G (IgG) solution to the PLL/ATP system. Using the knowledge from the phase diagram, we realized the formation of IgG/PLL droplets in a pharmaceutically required IgG concentration of ca. 10 mg/mL. Thus, this study provides guidance for using the phase diagram to analyze and utilize LLPS.
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Inmunoglobulina G , Polilisina , Humanos , Inmunoglobulina G/química , Adenosina TrifosfatoRESUMEN
Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.
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Industrias , L-Lactato Deshidrogenasa , Catálisis , Lisina , Poli A , PolímerosRESUMEN
Nanotechnology is a critical tool to manipulate the sophisticated behavior of biological structures and has provided new research fields. Liquid-liquid phase-separated (LLPS) droplets gather attention as basic reaction fields in a living cell. Droplets play critical roles in regulating protein behavior, including enzyme compartmentalization, stress response, and disease pathogenesis. The dynamic manipulation of LLPS droplet formation/deformation has become a crucial target in nanobiotechnology. However, the development of nanodevices specifically designed for this purpose remains a challenge. Therefore, this study presents butterfly-shaped gold nanobutterflies (GNBs) as novel nanodevices for manipulating LLPS droplet dynamics. The growth process of the GNBs is analyzed via time-lapse electroscopic imaging, time-lapse spectroscopy, and additives assays. Interestingly, GNBs demonstrate the ability to induce LLPS droplet formation in systems such as adenosine triphosphate/poly-l-lysine and human immunoglobulin G, whereas spherical and rod-shaped gold nanoparticles exhibit no such capability. This indicates that the GNB concave surface interacts with the droplet precursors facilitating the LLPS droplet formation. Near-infrared-laser irradiation applied to GNBs enables on-demand deformation of the droplets through localized heat effects. GNB regulates the enzymatic reaction of lysozymes. The innovative design of GNBs presents a promising strategy for manipulating LLPS dynamics and offers exciting prospects for future research.
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Oro , Nanopartículas del Metal , Humanos , ProteínasRESUMEN
Elucidating the link between amyloid fibril formation and liquid-liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective.
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Amiloide , Priones , Humanos , Amiloide/metabolismoRESUMEN
Complex coacervates have received increasing attention due to their use as simple models of membrane-less organelles and microcapsule platforms. The incorporation of proteins into complex coacervates is recognized as a crucial event that enables understanding of membrane-less organelles in cells and controlling microcapsules. Here, we investigated the incorporation of proteins into complex coacervates with a focus on the progress of the incorporation process. This stands in contrast to most previous studies, which have been focused the endpoint of the incorporation process. For that purpose, client proteins, i.e., lysozyme, ovalbumin, and pyruvate oxidase, were mixed with complex coacervate scaffolds consisting of two polyelectrolytes, i.e., the positively charged poly(diallyldimethylammonium chloride) and the negatively charged carboxymethyl dextran sodium salt, and the process was studied. Spectroscopic analysis and microscopic imaging demonstrated that electrostatic factors are the primary driving force of the incorporation of the client proteins into the complex coacervate scaffolds. Moreover, we discovered the formation of multi-phase droplets when a charged protein was incorporated into a complex coacervate whose surface was charged oppositely relative to that of the protein. The droplets inside the complex coacervates were found to be the diluted phase trapped as internal vacuoles. These findings provide fundamental insight into the temporal changes at the droplet interface during the incorporation of proteins into complex coacervates. This knowledge will facilitate the understanding of biological events associated with membrane-less organelles and will contribute to the industrial development of the use of microcapsules.
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Orgánulos , Proteínas , Humanos , Cápsulas , Proteínas/química , PolielectrolitosRESUMEN
Immobilized metal ion affinity chromatography (IMAC) is one of the most common purification techniques for histidine-tagged proteins (His-tagged proteins). IMAC enables the purification of His-tagged proteins at high purity on the basis of coordination bonds between His-tags and metal ions (such as Ni2+, Co2+, and Cu2+) immobilized on the matrices in columns. However, IMAC requires low-pH solutions or high-concentration imidazole solutions for eluting His-tagged proteins, which can affect protein conformation and activity. The present study provides a His-tagged protein purification method using zirconia particles modified with phosphate groups. This method is based on the electrostatic attractions between a His-tag moiety of proteins and phosphate groups on the zirconia particles; this method requires only high-concentration salt solutions at pH 7.0 for eluting the proteins. A column packed with phosphate-modified zirconia particles was demonstrated to enable the purification of two model His-tagged proteins-His-tagged green fluorescent protein and His-tagged alkaline phosphatase fused with maltose binding protein. Thus, this chromatography method is useful for purifying His-tagged proteins without any pH stress or additives. Additionally, because of the mechanical properties of the zirconia particles, this technique enables high-performance purification at a high flow rate.
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Histidina , Fosfatos , Histidina/química , Indicadores y Reactivos , Cromatografía de Afinidad/métodos , Proteínas RecombinantesRESUMEN
High-performance and cost-effective purification is necessary for the development of antibody drugs. This study found that nanoparticles of zirconia modified with phosphate groups selectively adsorb immunoglobulin G (IgG) antibodies against serum proteins with high adsorption capacity. The IgG antibodies collected from the zirconia nanoparticle surfaces retain their molecular conformation. Importantly, zirconia nanoparticles have the highest affinity for human IgG antibodies among tested mammalian IgG antibodies. The affinity for human IgG subclasses is in the order IgG3 > IgG1 > IgG2, which contrasts with a conventional ligand (Protein A) that has a lower affinity for IgG3. Because zirconia nanoparticles are chemically and mechanically stable, they can be utilized for the purification of antibody drugs not only in batch methods but also in chromatography as a process upstream or downstream of Protein A chromatography and even as an alternative process.
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Inmunoglobulina G , Fosfatos , Animales , Humanos , Adsorción , MamíferosRESUMEN
Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.
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Colorantes Fluorescentes , Orgánulos , Fluorescencia , Viscosidad , LisosomasRESUMEN
This study aimed to improve the emulsifying properties of commercial soy protein isolates (CSPIs). CSPIs were thermally denatured without additives (CSPI_H) and with arginine (CSPI_A), urea (CSPI_U), and guanidine hydrochloride (CSPI_G), which improve protein solubility to prevent aggregation. These additives were removed by dialysis, and the samples were lyophilized. CSPI_A resulted in high emulsifying properties. FT-IR analysis showed that the ß-sheet content in CSPI_A was reduced compared to that of untreated CSPI (CSPI_F). Fluorescence analysis showed that the tryptophan-derived emission peak of CSPI_A shifted between CSPI_F and CSPI_H which was exposed to hydrophobic amino acid chains with aggregation. As a result, the structure of CSPI_A became moderately unfolded and exposed the hydrophobic amino acid chains without aggregation. The CSPI_A solution had a more reduced oil-water interface tension than other CSPIs. These results support that CSPI_A attaches efficiently to the oil-water interface and produces small, less flocculated emulsions.
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Cell surface protein B (CspB) fusion proteins can undergo reversible pH-responsive precipitation-redissolution. A pH-responsive precipitation-redissolution of CspB tag purification (pPRCP) method was established for protein purification using this property. However, the mechanism of the pH-responsive precipitation of CspB fusion proteins is unknown, which has made it difficult to set process parameters for pPRCP. In this study, we investigated the mechanism of the pH-responsive precipitation of CspB fusion proteins using CspB50TEV-Teriparatide (CspB-teri) as a model. As expected, CspB-Teri was reversibly precipitated at acidic pH. By contrast, CspB-Teri was not precipitated under unfolding conditions induced by trifluoroethanol, urea, or guanidine hydrochloride, even at acidic pH. The conformation of CspB-Teri changed to a ß-sheet-rich structure as the pH decreased, followed by the formation of intermolecular interactions, which caused precipitation. The particle size of the CspB-Teri precipitate increased in a protein concentration-dependent manner. These results indicated that the pH-responsive precipitation of CspB-Teri is triggered by the formation of a ß-sheet structure in response to decreasing pH, and the growth of the precipitate particles occurred through intermolecular interactions.
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The assembly state of enzymes is gaining interest as a mechanism for regulating the function of enzymes in living cells. One of the current topics in enzymology is the relationship between enzyme activity and the assembly state due to liquid-liquid phase separation. In this study, we demonstrated enzyme activation via the formation of enzyme assemblies using L-lactate oxidase (LOX). LOX formed hundreds of nanometer-scale assemblies with poly-L-lysine (PLL). In the presence of ammonium sulfate, the LOX-PLL clusters formed micrometer-scale liquid droplets. The enzyme activities of LOX in clusters and droplets were one order of magnitude higher than those in the dispersed state, owing to a decrease in KM and an increase in kcat. Moreover, the clusters exhibited a higher activation effect than the droplets. In addition, the conformation of LOX changed in the clusters, resulting in increased enzyme activation. Understanding enzyme activation and assembly states provides important information regarding enzyme function in living cells, in addition to biotechnology applications.
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Oxigenasas de Función Mixta , Oxidorreductasas , Lisina , Proteína-Lisina 6-OxidasaRESUMEN
Uncovering the sequence-encoded molecular grammar that governs the liquid-liquid phase separation (LLPS) of proteins is a crucial issue to understand dynamic compartmentalization in living cells and the emergence of protocells. Here, we present a model LLPS system that is induced by electrostatic interactions between anionic nucleic acids and cationic oligolysine peptides modified with 12 different non-ionic amino acids, with the aim of creating an index of "phase-separation propensity" that represents the contribution of non-ionic amino acids to LLPS. Based on turbidimetric titrations and microscopic observations, the lower critical peptide concentrations where LLPS occurs (Ccrit) were determined for each peptide. A correlation analysis between these values and known amino acid indices unexpectedly showed that eight non-ionic amino acids inhibit the generation of LLPS, whereby the extent of inhibition increases with increasing hydrophobicity of the amino acids. However, three aromatic amino acids deviate from this trend and rather markedly promote LLPS despite their high hydrophobicity. A comparison with double-stranded DNA and polyacrylic acid revealed that this is primarily due to interactions with DNA nucleobases. Our approach to quantify the contribution of non-ionic amino acids can be expected to help to provide a more accurate description and prediction of the LLPS propensity of peptides/proteins.
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Aminoácidos , ADN , PéptidosRESUMEN
We present a pattern-recognition-based sensor that targets cell-derived components in culture media and evaluates cultured cells without damaging them. An array sensor made of a single-polymer probe was employed to obtain fluorescence response patterns of the analyte media, allowing successful identification of the type and state of the cells via multivariate analysis.
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Polímeros , Células Cultivadas , Medios de Cultivo , Análisis MultivarianteRESUMEN
Caries sensitivity varies between the two strains of inbred mice, BALB/cA has high sensitivity and C3H/HeN has low sensitivity. One potential reason seems to be a difference in pellicle-forming saliva protein composition. Here, we performed a proteomic analysis in order to identify differences of hydroxyapatite (HAP) adsorbed saliva proteins between these two mouse strains. HAP column chromatography revealed twice the quantity of high-affinity saliva proteins in C3H/HeN compared to BALB/cA. One- and two-dimensional electrophoresis showed 2 bands/spots with deviating migration. They were identified as murine carbonic anhydrase VI (CAVI) by peptide mass fingerprinting and confirmed with western blotting using a specific polyclonal antibody. Total RNA from the salivary glands of both mouse strains, PCR amplification of cDNA with a CAVI specific primer, and sequence analysis revealed one different base in codon 96, resulting in one different amino acid. Glyco-chains of CAVI deviate in one N-glycan, confirmed by mass analysis. CAVI activity was estimated from distinct circular dichroism spectra of the molecules and found higher in C3H/HeN mice. In summary, the CAVI composition of BALB/cA and C3H/HeN differs in one amino acid and a glyco-chain modification. Further, saliva from caries resistant C3H/HeN mice displayed higher CAVI activity and also overall hydroxyapatite adsorption, suggesting a relationship with caries susceptibility.
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Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals-Fe(II), Cu(II) and Ni(II)-on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.
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Histidina , Péptidos , Cationes , Cromatografía de Afinidad/métodos , Compuestos Ferrosos , Histidina/químicaRESUMEN
The affinity between amino acid and water is important for understanding how proteins behave in aqueous solutions. For example, the hydrophobicity of amino acid side chains determines a protein's solubility. However, the affinity of amino acid side chains in amino acid solvents should be determined in order to understand the propensity of protein condensates induced by multivalent amino acid interactions. Here we measured the transfer free energy of amino acid side chains (ΔGSC) from water to amino acid solvents. The ΔGSC of aromatic amino acids showed a different value depending on the type and the pH of amino acid solvent. Interestingly, the propensity of ΔGSC was completely different from the hydrophobicity of amino acids. This indicate that the ΔGSC describes the affinity between amino acid side chains involving the existence of water. The ΔGSC is a significant parameter for understanding whether amino acid side chains prefer bulk or protein condensate.