High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (ΔF/F = 15 to 30 in vitro), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an ex vivo preparation of Drosophila brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.

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.

Affinity of aromatic amino acid side chains in amino acid solvents.

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.

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.

Classification of protein solubilizing solutes by fluorescence assay.

Aromatic interaction plays a crucial role in controlling protein interaction by additives. Here we investigated the interaction of protein salting-in (solubilizing) additives with tryptophan (Trp), tyrosine (Tyr), indole, and proteins based on their fluorescence spectra. Five salting-in additives, i.e., arginine (Arg), urea, guanidine (Gdn), ethylene glycol (EG), and magnesium chloride (MgCl2), showed different effects on the fluorescence properties of Trp and Tyr. Arg significantly reduced fluorescence intensity of Trp and Tyr, as was the case for glycine to a lesser extent. MgCl2 and calcium chloride (CaCl2) showed little effect on the aromatic fluorescence spectra. Gdn also showed little effect on the aromatic fluorescence spectra of Trp and Tyr even at high concentrations. EG increased the aromatic fluorescence intensity of Trp and Tyr with blue-shifted emission wavelength. Urea enhanced fluorescence of Trp and Tyr without altering emission wavelength. These results indicate that the protein solubilizing additives interact with aromatic groups differently.

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution.

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid-liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.

Solubility Parameters of Amino Acids on Liquid-Liquid Phase Separation and Aggregation of Proteins.

The solution properties of amino acids determine the folding, aggregation, and liquid-liquid phase separation (LLPS) behaviors of proteins. Various indices of amino acids, such as solubility, hydropathy, and conformational parameter, describe the behaviors of protein folding and solubility both in vitro and in vivo. However, understanding the propensity of LLPS and aggregation is difficult due to the multiple interactions among different amino acids. Here, the solubilities of aromatic amino acids (SAs) were investigated in solution containing 20 types of amino acids as amino acid solvents. The parameters of SAs in amino acid solvents (PSASs) were varied and dependent on the type of the solvent. Specifically, Tyr and Trp had the highest positive values while Glu and Asp had the lowest. The PSAS values represent soluble and insoluble interactions, which collectively are the driving force underlying the formation of droplets and aggregates. Interestingly, the PSAS of a soluble solvent reflected the affinity between amino acids and aromatic rings, while that of an insoluble solvent reflected the affinity between amino acids and water. These findings suggest that the PSAS can distinguish amino acids that contribute to droplet and aggregate formation, and provide a deeper understanding of LLPS and aggregation of proteins.

Aromatic interaction of hydantoin compounds leads to virucidal activities.

Virus inactivation or disinfection is the first line of protection against virus infection. Here, we report for the first time the virus inactivation (virucidal) activities of hydantoin and its derivative, 1-methylhydantoin against enveloped herpes simplex virus type-1. These hydantoin compounds showed favorable interaction with aromatic amino acids, similarly to arginine hydrochloride also exhibiting aromatic interaction and virucidal activities on the same virus. Among them, 1-methylhydantoin demonstrated a greater virucidal activity. Solubility measurements in organic solvents and salting-out salt solutions showed that 1-methylhydantoin is more hydrophobic than others, suggesting that the hydrophobic nature and aromatic interaction play a role in interaction with viral proteins and thereby virucidal activity.

Glass-like protein condensate for the long-term storage of proteins.

Long-term storage of proteins at ambient temperature is required for applications in pharmaceutics and biotechnology. Lyophilization is a versatile approach for stabilizing proteins at ambient temperature, although its freezing and drying processes negatively affect the protein structure. In this study, we show a glass-like protein condensate (GLPC) as a new method for protein stabilization at ambient temperature. Various protein types, including immunoglobulin G, gamma globulin, albumin, and chymotrypsin, formed a glassy state during ultracentrifugation and natural drying, while proteins that tend to crystalize, such as hen egg-white lysozyme, did not. The GLPCs were characterized by a transparent solid state, similar to a dry glass ball. Importantly, the GLPCs were dissolved easily in saline solution at a physiological concentration, thereby retaining their native structures and functions. The GLPCs preserved their native structures even after 1 year of incubation at ambient temperature. These results provide a framework for the development of protein preservation methods at ambient temperature other than lyophilization.

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.

Effects of allantoin and dimethyl sulfoxide on the thermal aggregation of lysozyme.

Allantoin is used to suppress protein aggregation without decreasing the melting temperature. However, the solubility of allantoin in water or buffer solutions is too low (approximately 30 mM at ambient temperature) to be used as a protein aggregation suppressor in various situations. Here we show that a high-concentration solution of allantoin in neat dimethyl sulfoxide (DMSO) is useful as a stock solution for the additive that controls protein aggregation. The allantoin concentration from 10 to 100 mM in 10% (v/v) DMSO significantly suppressed the thermal aggregation of hen egg white lysozyme as a model protein, with increasing allantoin concentration. The residual activity of lysozyme in 10% DMSO and 100 mM allantoin after heating at 90 °C for 10 min was increased >3-fold compared to that without allantoin. Thus, it was concluded that allantoin in DMSO is an effective stock solution for practical application in enhancing the recovery of enzymatic activity and suppressing the formation of small aggregate of protein.

Arginine suppresses opalescence and liquid-liquid phase separation in IgG solutions.

Antibody formulation often necessitates the protein concentration to be increased above 100 mg/ml, because of the large therapeutic doses of antibodies required and the volume limitations of subcutaneous injections. However, high concentrations of antibody lead to opalescent states in solution, resulting in safety and application problems. In this study, we investigated the effect of additives on opalescence in IgG solutions. Arginine (Arg) was observed to most effectively suppress opalescence in IgG solutions among the additives tested, which included guanidine hydrochloride, NaCl, and other amino acids. Moreover, Arg also suppressed liquid-liquid phase separation (LLPS) of highly concentrated IgG solutions during incubation at low temperature. Comparative analysis showed that the effects of Arg on opalescence and LLPS in IgG solutions result from its unique structure, which comprises an amino acid main chain, a guanidinium group, and a counter ion. These results indicate that Arg has high potency as an excipient in antibody drug formulations for the suppression of opalescence and LLPS as well as protein aggregation.

Allantoin and hydantoin as new protein aggregation suppressors.

Allantoin is widely used in pharmaceutical and cosmetic products, and is composed of a hydantoin ring and a ureido group. Recent reports showed that allantoin suppresses thermal aggregation of hen egg white lysozyme (LYZ). However, structural insight into the properties of allantoin on protein aggregation and whether allantoin controls the aggregation of other proteins under different stress conditions remain unclear. Here we studied the structural properties of allantoin in terms of its effects on protein aggregation by comparing allantoin with urea and hydantoin. Furthermore, we analyzed the effects of allantoin and its derivatives on the aggregation of LYZ, carbonic anhydrase from bovine erythrocytes (BCA), albumin from chicken egg white (OVA), and immunoglobulin G (IgG) by various stresses in comparison with arginine. These four proteins are widely different in charged state and molecular size. Allantoin suppressed the aggregation and inactivation of LYZ comparing to arginine without affecting the melting temperature of proteins, and was responsible for the slightly improved formation of soluble oligomers and insoluble aggregates of IgG with thermal and acidic stresses. In contrast, hydantoin increased the solubility of aromatic amino acids more effectively than arginine and allantoin. The structural properties underlying the observed effects of allantoin as an aggregation suppressor include hydrophobic interactions between hydantoin moiety and aromatic ring on the surface of proteins, which is reflected on the difference between allantoin and arginine. These results show that the backbone of hydantoin ring may be a new category of additives for development of small aggregation suppressors.

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.