Cationic polyelectrolytes prevent the aggregation of l-lactate dehydrogenase under unstable conditions.

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.

Activation of oxidoreductases by the formation of enzyme assembly.

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.

Transient formation of multi-phase droplets caused by the addition of a folded protein into complex coacervates with an oppositely charged surface relative to the protein.

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.

Activation of L-lactate oxidase by the formation of enzyme assemblies through liquid-liquid phase separation.

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.

Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns.

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.

Dynamic behavior of liquid droplets with enzyme compartmentalization triggered by sequential glycolytic enzyme reactions.

Dynamic droplet formation via liquid-liquid phase separation (LLPS) is believed to be involved in the regulation of various biological processes. Here, a model LLPS system coupled with a sequential glycolytic enzymatic reaction was developed to reproduce the dynamic control of liquid droplets; (i) the droplets, which consist of poly-L-lysine and nucleotides, compartmentalize two different enzymes (hexokinase and glucose-6-phosphate dehydrogenase) individually, accelerating the overall reaction, and (ii) each enzymatic reaction triggers the formation, dissolution and long-term retention of the droplets by converting the scaffold nucleotides. This model system will offer a new aspect of enzymes associated with LLPS in living cells.

Insight into the protein salting-in mechanism of arginine, magnesium chloride and ethylene glycol: Solvent interaction with aromatic solutes.

Key factors in the salting-in effects on proteins of additives are their interactions with aromatic groups. We studied the interaction of four aromatic solutes, benzyl alcohol (BA), phenol, 4-hydroxybenzyl alcohol (4-HBA) and methyl gallate (MG), with different salting-in additives, arginine hydrochloride (ArgHCl), magnesium chloride (MgCl2), ethylene glycol (EG), and guanidine hydrochloride (GdnHCl) using solubility measurements. We used sodium chloride (NaCl) as a control. MgCl2 decreased the solubility of the four aromatic solutes with weak solute dependence. In contrast, ArgHCl, GdnHCl, and EG increased the solubility of four aromatic solutes with a similar solute dependence. Their salting-in effects were weaker on BA and 4-HBA and stronger on phenol and MG. These results indicate that attached groups alter the aromatic properties, affecting the interactions between the benzene ring and these three additives. More importantly, the observed results demonstrate that the salting-in mechanism is different between MgCl2, EG and ArgHCl, which should play a role in their effects on protein solubility.

Effect of additives on liquid droplets and aggregates of proteins.

This review briefly summarizes the effect of additives on the formation of liquid droplets and aggregates of proteins. Proteins have the property of forming liquid droplets and aggregates both in vivo and in vitro. The liquid droplets of proteins are mainly stabilized by electrostatic and cation-π interactions, whereas the amorphous aggregates are mainly stabilized by hydrophobic interactions. Crowders usually stabilize liquid droplets, whereas ions and hexandiols destabilize the droplets. Additives such as kosmotropes, sugars, osmolytes, and crowders promote the formation of amorphous aggregates, whereas additives such as arginine and chaotropes can prevent the formation of amorphous aggregates. Further, amyloid has a different mechanism for its formation from amorphous aggregates because it is primarily stabilized by a cross-ß structure. These systematic analyses of additives will provide clues to controlling protein aggregations and will aid the true understanding of the transition of proteins from liquid droplets and aggregates.

The binding affinity of uncharged aromatic solutes for negatively charged resins is enhanced by cations via cation-π interactions: The case of sodium ion and arginine.

Salt solutions are widely used as eluents for ion-exchange chromatography. In general, salts reduce the retention of applied solutes on ion-exchange columns via electrostatic screening effects. The reverse phenomenon, namely, salt-enhanced retention, has not been reported. Here, we report that cations, including arginine, guanidine and sodium ions, enhance the retention of uncharged aromatic solutes on a cation-exchange resin, i.e., a negatively charged resin, with carboxyl groups, where we used alkyl gallates as model uncharged aromatic solutes and a carboxymethyl agarose gel (CM Sepharose) as a model negatively charged resin. Enhancement of retention was observed at concentrations of tens of millimolar of the salts, in which arginine hydrochloride was more effective than guanidinium salts and NaCl. Similar trends were observed for other phenolic compounds, including phenol and 4-hydroxybenzyl alcohol. Molecular dynamics simulations showed that the binding free energy between the alkyl gallate molecule and the CM Sepharose resin ligand molecule increased with increasing salt concentration. The increase in binding free energy caused by the salts was accounted for by the binding of the salt cations to the aromatic moiety of the alkyl gallate via cation-π interactions, leading to attenuation of intrinsic repulsive interactions between the ligand carboxyl group and the alkyl gallate aromatic moiety. Therefore, the salt-enhanced retention of the uncharged aromatic solutes on the negatively charged resins was ascribable to the increase in binding free energy induced by the cation-π interactions. This unique reverse phenomenon of the effect of salts on solute retention indicates the importance of cation-π interactions in ion-exchange chromatography. This phenomenon can be used for selective chromatographic separation of aromatic solutes, including organic solutes, proteins and nucleic acids.

Cytosolic Transport of Nanoparticles through Pressurized Plasma Membranes for Molecular Delivery and Amplification of Intracellular Fluorescence.

Transporting nanoparticles into live cells is important for drug delivery and other related applications. We found that cells exposed to hypoosmotic pressures can internalize substantial quantities of gold nanoparticles. Importantly, these nanoparticles can circumvent normal intracellular traffic and be transported directly into the cytosol, without the need for surface functionalization. In contrast, nanoparticles endocytosed at physiological osmolality are segregated inside endocytic organelles and are not able to reach the cytosol. Cytosolic internalization was observed for nanoparticles of various sizes and materials, with minimal short- or long-term damage induced by the internalized particles. Thus, our strategy can be used as a delivery platform for a range of applications from therapeutics to medical imaging. As examples, we demonstrated rapid delivery of membrane-impermeable molecules to the cytosol by using nanoparticles as carriers and the use of nanoparticles assembled within the cytosol as plasmonic nanoantenna to enhance intracellular fluorescence. We propose a model for the mechanisms behind nanoparticle internalization through pressurized plasma membranes via the release of lateral pressures. Such characterizations may constitute a foundation for developing new technologies, including nanoparticle-based drug delivery.