Detection of Fibril Nucleation in Micrometer-Sized Protein Condensates and Suppression of Sup35NM Fibril Nucleation by Liquid-Liquid Phase Separation.

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.

Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection.

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.

Synthesis of Butterfly-Like Shaped Gold Nanomaterial: For the Regulation of Liquid-Liquid Phase-Separated Biomacromolecule Droplets.

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.

Phase-Diagram Observation of Liquid-Liquid Phase Separation in the Poly(l-lysine)/ATP System and a Proposal for Diagram-Based Application Strategy.

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.

Phase-Separation Propensity of Non-ionic Amino Acids in Peptide-Based Complex Coacervation Systems.

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.

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.

Opalescence Arising from Network Assembly in Antibody Solution.

Opalescence of therapeutic antibody solutions is one of the concerns in drug formulation. However, the mechanistic insights into the opalescence of antibody solutions remain unclear. Here, we investigated the assembly states of antibody molecules as a function of antibody concentration. The solutions of bovine gamma globulin and human immunoglobulin G at around 100 mg/mL showed the formation of submicron-scale network assemblies. The network assembly resulted in the appearance of opalescence with a transparent blue color without the precipitates of antibodies. Furthermore, the addition of trehalose and arginine, previously known to act as protein stabilizers and protein aggregation suppressors, was able to suppress the opalescence arising from the network assembly. These results will provide an important information for evaluating and improving protein formulations.

Solution design to extend the pH range of the pH-responsive precipitation of a CspB fusion protein.

Cell surface protein B (CspB) from Corynebacterium glutamicum has been developed as a reversible pH-responsive tag for protein purification. CspB fusion proteins precipitate at acidic pH, after that they completely dissolve at neutral pH. This property has been used in a non-chromatographic protein purification method named pH-responsive Precipitation-Redissolution of CspB tag Purification (pPRCP). However, it is difficult to apply pPRCP to proteins that are unstable under acidic conditions. In an effort to shift the precipitation pH to a milder range, we investigated the solution conditions of CspB-fused Teriparatide (CspB50TEV-Teriparatide) during the process of pH-responsive precipitation using pPRCP. The purified CspB50TEV-Teriparatide in buffer without additives precipitated at pH 5.3. By contrast, CspB50TEV-Teriparatide in buffer with 0.5 M Na2SO4 precipitated at pH 6.6 because of the kosmotropic effect. Interestingly, the pH at which precipitation occurred was independent of the protein concentration. The precipitated CspB50TEV-Teriparatide was fully redissolved at above pH 8.0 in the presence or absence of salt. The discovery that proteins can be precipitated at a mild pH will allow pPRCP to be applied to acid-sensitive proteins.

Quadruplex Folding Promotes the Condensation of Linker Histones and DNAs via Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that, in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with a high charge density. Given that DNA quadruplex structures are well-documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.

Arginine is a disease modifier for polyQ disease models that stabilizes polyQ protein conformation.

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal ß-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. Here we identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. We first screened representative chemical chaperones using an in vitro polyQ aggregation assay, and identified arginine as a potent polyQ aggregation inhibitor. Our in vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic ß-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.

Selective separation method of aggregates from IgG solution by aqueous two-phase system.

Aggregation of immunoglobulin G (IgG) is a serious concern that results in immunogenicity in pharmaceutical applications. Removal of the small and soluble aggregates in protein solutions through a simple method remains challenging. Here we show that an aqueous two-phase system (ATPS) can be used for the elimination of soluble aggregates from IgG solution. Polyethylene glycol (PEG) and dextran (DEX) were selected as components of the ATPS. As expected, IgG monomers were partitioned into the top or bottom phases of ATPS. Interestingly, almost all the small and soluble aggregates of IgG were extracted to the interface between top and bottom phases, rather than in the liquid phases. The partitioning of monomers and aggregates of IgG can be attributed to the solubility of these protein states in PEG and DEX. Thus, ATPS using PEG and DEX can be employed for the simple removal method of soluble aggregates from IgG solution.

Non-chromatographic purification of Teriparatide with a pH-responsive CspB tag.

Cell surface protein B (CspB) from Corynebacterium glutamicum is used as a pH-responsive peptide tag to enable a simple solid-liquid separation method for isolating a CspB fusion protein. Here we demonstrate the first application of a CspB tag for the purification of Teriparatide, which is a biologic drug that is prescribed for osteoporosis. The Teriparatide was constructed as CspB50TEV-Teriparatide, comprising 50 amino acid residues of CspB, the cleavage site of TEV protease, and Teriparatide. CspB50TEV-Teriparatide was expressed in a culture supernatant by C. glutamicum secretion system at 3.0 g/L (equivalent to approximately 1.2 g/L Teriparatide). The CspB50TEV-Teriparatide was precipitated by reducing the pH of the culture supernatant, and the precipitate was then dissolved in a neutral buffer. A TEV protease treatment was applied to cleave the Teriparatide from the CspB50TEV-Teriparatide. Then, the remaining digested CspB50TEV, undigested CspB50TEV-Teriparatide, and TEV protease were precipitated in an acidic pH, whereas the soluble Teriparatide remained in the supernatant. The process had a yield of 96.5% and resulted in Teriparatide with a purity of 98.0% and productivity of 1.1 g/L of C. glutamicum culture. Thus, tag-free Teriparatide was successfully purified from the CspB fusion protein using only pH changes, centrifugation, and protease digestion without the need for chromatography. This versatile purification protocol is expected to be applicable to various proteins from laboratory to industrial scales.

Effect of additives on liquid droplet of protein-polyelectrolyte complex for high-concentration formulations.

Liquid droplets of protein-polyelectrolyte complexes (PPCs) have been developed as a new candidate for stabilization and concentration of protein drugs. However, it remains unclear whether additives affect the precipitation and redissolution yields of PPCs. In the present study, we investigated the PPC formation of human immunoglobulin G (IgG) and poly-L-glutamic acid (polyE) in the presence of various additives that have diverse effects, such as protein stabilization. Alcohols, including ethanol, successfully increased the PPC precipitation yield to over 90%, and the PPCs formed were completely redissolved at physiological ionic strength. However, poly(ethylene glycol), sugars, and amino acids did not improve the precipitation and redissolution yields of PPCs over those observed when no additives were included. Circular dichroism spectrometry showed that the secondary structure of polyE as well as electrostatic interactions play important roles in increasing the PPC precipitation yield when ethanol is used as an additive. The maximum concentration of IgG reached 100 mg/ml with the use of ethanol, which was 15% higher efficiency of the protein yield after precipitation and redissolution than that in the absence of additives. Thus, the addition of a small amount of ethanol is effective for the concentration and stabilization of precipitated PPCs containing IgG formulations.

Specific solubilization of impurities in culture media: Arg solution improves purification of pH-responsive tag CspB50 with Teriparatide.

Protein purification using non-chromatographic methods is a simple technique that avoids costly resin. Recently, a cell surface protein B (CspB) tag has been developed for a pH-responsive tag for protein purification by solid-liquid separation. Proteins fused with the CspB tag show reversible insolubilization at acidic pH that can be used in solid-liquid separation for protein purification. However, brown-color impurities from co-precipitation hamper further analysis of the target proteins. In this study, we investigated the effect of additives on the co-precipitation of CspB-tagged Teriparatide (CspB50TEV-Teriparatide) expressed in Corynebacterium glutamicum and associated impurities. Arginine (Arg) at 1.0 M was found to be the most effective additive for removing impurities, particularly carotenoids and nucleic acids. Furthermore, all impurities detected in the fluorescence and absorbance spectra were successfully removed by the repetition of precipitation-redissolution in the Arg solution. The precipitation yield of the CspB50TEV-Teriparatide did not change with the addition of Arg and the repetition of the precipitation-redissolution process. Collectively, our findings indicate that the specific desorption of π-electron rich compounds by Arg may be useful in conjunction with the pH-responsive CspB tag for solid-liquid protein purification.

A new pH-responsive peptide tag for protein purification.

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH.

One-Step Identification of Antibody Degradation Pathways Using Fluorescence Signatures Generated by Cross-Reactive DNA-Based Arrays.

Therapeutic antibodies are prone to degradation via a variety of pathways during each stage of the manufacturing process. Hence, a low-cost, rapid, and broadly applicable tool that is able to identify when and how antibodies degrade would be highly desirable to control the quality of therapeutic antibody products. With this goal in mind, we have developed signature-based sensing system to discriminate differently degraded therapeutic antibodies. The use of arrays consisting of conjugates between nanographene oxide and fluorophore-modified single-stranded DNAs under acidic pH conditions generated unique fluorescence signatures for each state of the antibodies. Multivariate analyses of the thus obtained signatures allowed identifying (i) common features of native, denatured, and visibly aggregated antibodies, (ii) complicated degradation pathways of therapeutic omalizumab upon time-course heat-treatment, and (iii) the individual compositions of differently degraded omalizumab mixtures. As the signature-based sensing has the potential to identify a broad range of degraded antibodies formed by different kinds of realistic stress types, this system may serve as the basis for high-throughput assays for the screening of antibody manufacturing processes.

A study of the small-molecule system used to investigate the effect of arginine on antibody elution in hydrophobic charge-induction chromatography.

Hydrophobic charge-induction chromatography (HCIC) using 4-mercaptoethylpyridine (4-MEP) as the ligand is used to purify antibodies. The 4-MEP resin ligand has high affinity for antibodies, which makes it difficult to optimize the elution conditions. Recent studies showed that arginine is effective at eluting and purifying antibodies using the HCIC with 4-MEP. In the present study, we investigated the mechanism of the action of arginine on the interaction between butyl gallate (BG) and the 4-MEP resin as a model system for protein-4-MEP interactions. Equilibrium adsorption experiments showed that arginine has a significant effect on the desorption of BG from the 4-MEP resin and, in fact, is found to exhibit a greater effectiveness than guanidine and urea, which are known denaturants. The calculated binding free energy between a BG molecule and a 4-MEP resin ligand molecule using molecular dynamics simulations was qualitatively consistent with the experimental results. A principal component analysis of the simulations showed that arginine molecules intervene in the interaction between the BG and 4-MEP molecules at a distance of 8.5 Å by entering the space between the phenol and pyridine planes. The present results suggest that arginine has a unique mechanism of interaction with the phenol-pyridine system, which should be associated with the effects of arginine on the protein-4-MEP systems.

Salt effects on the picosecond dynamics of lysozyme hydration water investigated by terahertz time-domain spectroscopy and an insight into the Hofmeister series for protein stability and solubility.

The addition of salts into protein aqueous solutions causes changes in protein solubility and stability, whose ability is known to be ordered in the Hofmeister series. We investigated the effects of Hofmeister salts on the picosecond dynamics of water around a lysozyme molecule using terahertz time-domain spectroscopy. The change in the absorption coefficient for 200 mg mL(-1) lysozyme aqueous solution by the addition of salts was found to depend on the salts used, whereas that for pure water was almost independent of salts. From the difference in the salt concentration dependence for various salts, it has been found that chaotropic anions make the dynamics of water around the lysozyme molecule slower, whereas kosmotropic anions make the dynamics faster. The ability of an anion to slow down the water dynamics was found to have the following order: SCN(-) > Cl(-) > H2PO4(-) > NO3(-) ≈ SO4(2-). This result indicates that the effects of anions on the dynamics of water around the lysozyme molecule are the opposite of those for bulk water. This finding agrees with a prediction from a molecular model proposed by Collins [K. D. Collins, Methods, 2004, 34, 300]. The results presented here are compared with the results from preferential interaction studies and the results from sum frequency generation spectroscopy. These discussions have led to the conclusion that the picosecond dynamics of protein hydration water strongly contributes to protein stability, whereas electrostatic interactions between protein molecules contribute to protein solubility.

Liquid Chromatographic Analysis of the Interaction between Amino Acids and Aromatic Surfaces Using Single-Wall Carbon Nanotubes.

Proteins have nonspecific adsorption capacities for solid surfaces. Although the nonspecific adsorption capacities are generally understood to be related to the hydrophobicity or charge density of the surfaces, little is known at the amino acid level about the interaction between proteins and polyaromatic surfaces such as carbon nanotubes, which have recently been used for biotechnology applications. In this study, we investigated the interaction between proteinogenic amino acids and carbon nanotubes using high-performance liquid chromatography on silica matrices coated by single-wall carbon nanotubes (SWCNTs). Among the amino acids used in this study, tryptophan, tyrosine, and phenylalanine showed exceptional affinity for the matrices. The characteristic affinities of these amino acids were ascribed to their unique interactions with the large polyaromatic surfaces of the SWCNTs. These results are useful for understanding and controlling protein adsorption onto aromatic surfaces.

Specific decrease in solution viscosity of antibodies by arginine for therapeutic formulations.

Unacceptably high viscosity is observed in high protein concentration formulations due to extremely large therapeutic dose of antibodies and volume restriction of subcutaneous route of administration. Here, we show that a protein aggregation suppressor, arginine hydrochloride (ArgHCl), specifically decreases viscosity of antibody formulations. The viscosities of bovine gamma globulin (BGG) solution at 250 mg/mL and human gamma globulin (HGG) solution at 292 mg/mL at a physiological pH were too high for subcutaneous injections, but decreased to an acceptable level (below 50 cP) in the presence of 1,000 mM ArgHCl. ArgHCl also decreased the viscosity of BGG solution at acidic and alkaline pHs. Interestingly, ArgHCl decreased the viscosity of antibody solutions (BGG, HGG, and human immunoglobulin G) but not globular protein solutions (α-amylase and α-chymotrypsin). These results indicate not only high potency of ArgHCl as an excipient to decrease the solution viscosity of high concentration antibodies formulations but also specific interactions between ArgHCl and antibodies.