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1.
Biomacromolecules ; 24(2): 704-713, 2023 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-36640113

RESUMO

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.


Assuntos
Aminoácidos , DNA , Peptídeos
2.
Chem Commun (Camb) ; 58(96): 13317-13320, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36342014

RESUMO

The enzymatic luminescence reactions of fireflies are accelerated in the presence of biomolecular condensates comprising a positively charged peptide and ATP. We revealed that this acceleration is caused by the enrichment of reaction elements, local pH changes, and promotion of inhibitory intermediate dissociation, improving the bioluminescence quantum yield by approximately 10%.


Assuntos
Vaga-Lumes , Luciferases de Vaga-Lume , Animais , Vaga-Lumes/química , Condensados Biomoleculares , Luminescência
3.
J Biosci Bioeng ; 133(1): 17-24, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34629298

RESUMO

High-concentration and low-viscosity antibody formulations are necessary when administering these solutions subcutaneously (SC) due to limitations on injection volume. Here we show a method to decrease the viscosity of monoclonal antibody solution by protein-polyelectrolyte complex (PPC) with poly-l-glutamic acid (polyE). The viscosity of omalizumab solutions was 90 cP at the concentration of 150 mg/mL. In the presence of 20-50 mM polyE, the viscosity of PPC solution of 150 mg/mL omalizumab dramatically decreased below 10 cP due to the formation of crowded solution. The crowded state of PPC, named aggregated PPC (A-PPC), contained water droplets with a diameter of 10 µm or larger with low antibody concentrations. In the presence of 60 mM or more polyE, the omalizumab solution was transparent with the viscosity of 40 cP or less, named soluble PPC (S-PPC). More importantly, the solutions of both A-PPC and S-PPC were fully redissolved by the addition of phosphate saline buffer confirmed by secondary structure, the amount of aggregates, and binding activity to antigen.


Assuntos
Anticorpos Monoclonais , Composição de Medicamentos , Polieletrólitos , Estrutura Secundária de Proteína , Soluções , Viscosidade
4.
Front Cell Dev Biol ; 9: 710729, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34422830

RESUMO

Liquid-liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

5.
J Am Chem Soc ; 143(26): 9849-9857, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34152774

RESUMO

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.


Assuntos
DNA/química , Histonas/química , Sequência de Aminoácidos , Quadruplex G , Heterocromatina/química , Extração Líquido-Líquido , Ligação Proteica , Domínios Proteicos
6.
Anal Sci ; 37(5): 713-719, 2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-33518589

RESUMO

Pattern-recognition-based sensing has attracted attention as a promising alternative to conventional sensing methods that rely on selective recognition. Here, we report on novel strategy using chemical additives with the ability to modulate probe/analyte interactions to more easily construct pattern-recognition-based sensing systems for proteins and cells. The fluorescence of dansyl-modified cationic poly-L-lysine (PLL-Dnc) is enhanced upon binding to proteins in aqueous solution, while the addition of salts, inert polymers, or alcohols modulates the protein/PLL-Dnc interactions via a variety of mechanisms. Subsequent readout of the fluorescence changes produces response patterns that reflect the characteristics of the analytes. Multivariate analysis of the response patterns allowed for accurate identification of not only eight structurally similar albumin homologues, but also four mammalian cells. This strategy, which uses inexpensive and common additives, significantly improves the accessibility of pattern-recognition-based sensing, which will offer new opportunities for the detection of various bioanalytes.


Assuntos
Técnicas Biossensoriais , Polímeros , Animais , Fluorescência , Lisina , Proteínas
7.
Biophys Rev ; 12(2): 587-592, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32166611

RESUMO

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.

8.
ACS Appl Mater Interfaces ; 11(50): 47428-47436, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31747245

RESUMO

The detection of proteases and their complexes with inhibitor proteins is of great importance for diagnosis and medical-treatment applications. In this study, we report a fingerprint-based sensor using an array of single-stranded DNAs (ssDNAs) labeled with environment-responsive 3'-carboxytetramethylrhodamine (TAMRA) for the identification of proteases. Four TAMRA-modified ssDNAs with different sequences solubilized in two different buffer solutions were incorporated in an array that was capable of generating fluorescent fingerprints unique to the proteases through diverse cross-reactive interactions, allowing the discrimination of (i) 8 proteases and (ii) 12 different mixtures of trypsin and its inhibitor protein (α1-antitrypsin) by multivariate analysis. Constructing an array with TAMRA-modified DNA aptamers that bind to different sites of human thrombin provides fluorescence fingerprints that reflect a reduction of the exposed surface area of thrombin upon complexation with antithrombin III, even in the presence of human serum. We finally demonstrate the potential of hybridization with complementary DNAs as an effective means to easily double the fingerprint information for proteases. Our approach based on the cross-reactive capability of ssDNAs enables high-throughput fingerprint-based sensing that can be flexibly designed and easily constructed, not only for the identification of a variety of proteins including proteases but also for the evaluation of their complexation ability.


Assuntos
Técnicas Biossensoriais , Complexos Multiproteicos/isolamento & purificação , Peptídeo Hidrolases/isolamento & purificação , Trombina/química , Antitrombina III/química , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Humanos , Complexos Multiproteicos/química , Peptídeo Hidrolases/química , Ligação Proteica , Rodaminas/química , Tripsina/química , Tripsina/isolamento & purificação , alfa 1-Antitripsina/química , alfa 1-Antitripsina/isolamento & purificação
9.
J Chem Phys ; 150(6): 064903, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769990

RESUMO

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.

10.
Anal Sci ; 35(1): 99-102, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29806617

RESUMO

A differential array consisting of commercially available common fluorescent dyes was constructed for the identification of proteins and human cancer cells. Fluorescence of dyes was differently altered by mixing with proteins and human cancer cells, generating response patterns that are unique to the analytes. Linear discriminant analysis of the obtained patterns enabled the accurate identification of eight proteins and three human cancer cells. As this system can be easily prepared, it would offer a unique opportunity for array-based differential biosensing.


Assuntos
Técnicas Biossensoriais , Corantes Fluorescentes/química , Medições Luminescentes , Proteínas/análise , Proteínas/química , Análise Discriminante , Humanos , Peso Molecular , Análise de Componente Principal , Células Tumorais Cultivadas
11.
Curr Pharm Biotechnol ; 19(12): 946-955, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30514188

RESUMO

This review article presents the concepts of aggregates, coaggregates, and a liquid droplet of proteins and compares the concentrated states in the presence of small additives to control their formation and dissociation. The aggregates composed of single protein molecules result mainly from hydrophobic interactions between unfolded protein molecules. Thus, the aggregation of protein can be effectively suppressed by small additives that increase the solubility of hydrophobic solutes, typically arginine (Arg) and chaotropes. In contrast, coaggregation that is composed of two or more types of proteins results from both hydrophobic and attractive electrostatic interactions between even partially unfolded protein molecules. Accordingly, coaggregation is more controllable than simple aggregation using various types of small additives, such as ions and osmolytes, as well as Arg and chaotropes. The liquid droplets of proteins observed in living cells and a protein-polyelectrolyte complex (PPC) undergo liquid-liquid phase separation driven only by electrostatic interactions. Thus, the liquid droplets and PPCs are redissolved when the concentration of ions is increased. The properties of electrostatic or hydrophobic interactions, solid-like or liquid-like states, and apparently spherical and amorphous structures are simple but valuable criteria that can be used to control protein aggregation and condensation using small additives.


Assuntos
Lipídeos/química , Agregados Proteicos , Desdobramento de Proteína , Proteínas/química , Solventes/química , Animais , Arginina/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Transição de Fase , Polieletrólitos/química , Solubilidade , Soluções , Eletricidade Estática
12.
J Pharm Sci ; 107(10): 2713-2719, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29960025

RESUMO

The formulation of high-concentration protein solutions is a challenging issue for achieving subcutaneous administration. Previously, we developed a method of precipitation-redissolution using polyelectrolyte as a precipitant to produce protein solutions at high concentrations. However, the redissolution yield of proteins was insufficient. This study aims to optimize the solution conditions for practical applications by combining IgG and poly-l-(glutamic acid) (polyE). A systematic analysis of solution pH and polyE size conditions revealed that an acidic condition favors precipitation, whereas neutral pH values are more effective for the redissolution. We find that the optimal size for polyE ranged from 15,000 to 50,000. This slight modification in the procedure in comparison with previous studies increased the precipitation and redissolution yields to nearly 100%, without irreversible protein denaturation. The fully reversible IgG-polyE complex formed as a droplet structure, which is similar to a condensate of liquid-liquid phase separation. The droplet structure plays an indispensable role in the salt-induced, redissolved state, which is pertinent to the new application that takes advantage of the methods to produce highly concentrated protein solutions.


Assuntos
Preparações Farmacêuticas/química , Polieletrólitos/química , Proteínas/química , Precipitação Química , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Solubilidade
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