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 (Δ/ = 15 to 30 ), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities.

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.

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.

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.

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.

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.

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.

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 and . However, understanding the propensity of LLPS and aggregation is difficult due to the multiple interactions among different amino acids.

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.

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.

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.

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.

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.

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.

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.