RESUMEN
Proteinaceous liquid droplets, generated by liquid-liquid phase separation, function as membraneless compartments that are essential for diverse biological functions. Studies addressing droplet generation have used 1,6-hexanediol (1,6-HD) as a droplet-discerning agent owing to its capacity to induce droplet deformation. Despite the empirical utility of 1,6-HD, the mechanism underlying 1,6-HD-induced droplet deformation remains unknown. In this study, the solubilities of N-acetyl amino acid amides, which correspond to proteinogenic amino acid residues, were examined in the presence of 1,6-HD at 25 °C. Other solvents included ethanol, 1-propanol, and amides. Remarkably, 1,6-HD effectively solubilized hydrophobic species (particularly aromatic species) and exhibited reduced efficacy in solubilizing hydrophilic species and peptide bond moieties. These solubilizing effects are reflected in changes in protein solubility and structure. Specifically, 1,6-HD primarily targets the hydrophobic regions of a protein, increasing protein solubility without causing substantial structural changes. This solubilization mechanism is essential for elucidating the role of 1,6-HD as a droplet-discerning agent and recognizing its potential limitations.
Asunto(s)
Amidas , Aminoácidos , Glicoles , Solubilidad , Amidas/química , Solventes/química , Agua , ProteínasRESUMEN
Carbon nanobelts (CNBs) correspond to carbon nanotube (CNT) segments and are insoluble in most common aqueous solutions, posing challenges across diverse applications. In this study, [12] CNB, which corresponds to a (6,6) CNT segment, was solubilized by aliphatic surfactant micelles through host-guest complexation, which was confirmed by comprehensive analyses involving spectrophotometry, mass spectrometry, and molecular dynamics simulations. Through this solubilization, zero-Stokes shift emission of the CNB could occur, which could be ascribed to the symmetry-allowed transition. In contrast, CNB was insoluble in non-aliphatic surfactant solutions. The mechanism by which CNB is solubilized using aliphatic surfactants is completely distinct from that of the CNT dispersion mechanism. The present finding provides knowledge of the effectiveness of aliphatic compounds in solubilizing CNBs and their derivatives (carbon nanohoops), which show significant potential for various applications in aqueous systems, including biological applications.
RESUMEN
Immobilized metal ion affinity chromatography (IMAC) is one of the most common purification techniques for histidine-tagged proteins (His-tagged proteins). IMAC enables the purification of His-tagged proteins at high purity on the basis of coordination bonds between His-tags and metal ions (such as Ni2+, Co2+, and Cu2+) immobilized on the matrices in columns. However, IMAC requires low-pH solutions or high-concentration imidazole solutions for eluting His-tagged proteins, which can affect protein conformation and activity. The present study provides a His-tagged protein purification method using zirconia particles modified with phosphate groups. This method is based on the electrostatic attractions between a His-tag moiety of proteins and phosphate groups on the zirconia particles; this method requires only high-concentration salt solutions at pH 7.0 for eluting the proteins. A column packed with phosphate-modified zirconia particles was demonstrated to enable the purification of two model His-tagged proteins-His-tagged green fluorescent protein and His-tagged alkaline phosphatase fused with maltose binding protein. Thus, this chromatography method is useful for purifying His-tagged proteins without any pH stress or additives. Additionally, because of the mechanical properties of the zirconia particles, this technique enables high-performance purification at a high flow rate.
Asunto(s)
Histidina , Fosfatos , Histidina/química , Indicadores y Reactivos , Cromatografía de Afinidad/métodos , Proteínas RecombinantesRESUMEN
High-performance and cost-effective purification is necessary for the development of antibody drugs. This study found that nanoparticles of zirconia modified with phosphate groups selectively adsorb immunoglobulin G (IgG) antibodies against serum proteins with high adsorption capacity. The IgG antibodies collected from the zirconia nanoparticle surfaces retain their molecular conformation. Importantly, zirconia nanoparticles have the highest affinity for human IgG antibodies among tested mammalian IgG antibodies. The affinity for human IgG subclasses is in the order IgG3 > IgG1 > IgG2, which contrasts with a conventional ligand (Protein A) that has a lower affinity for IgG3. Because zirconia nanoparticles are chemically and mechanically stable, they can be utilized for the purification of antibody drugs not only in batch methods but also in chromatography as a process upstream or downstream of Protein A chromatography and even as an alternative process.
Asunto(s)
Inmunoglobulina G , Fosfatos , Animales , Humanos , Adsorción , MamíferosRESUMEN
Carbon nanotubes (CNTs) have adverse impacts on metabolism in biological systems. The impacts should be associated with interactions of the CNTs with coenzymes, such as nicotinamide adenine dinucleotide (NAD), because most metabolic processes are governed by coenzyme-dependent reactions. This study demonstrates that NAD molecules adsorb onto the CNT surface, leading to the formation of interfacial NAD layers-in other words, a coenzyme corona (coenzyme-based biomolecular corona). Coenzyme corona formation is accompanied by the oxidation of NAD at biological concentrations through electron transfer. Similar phenomena are observed for NAD derivatives. Molecular dynamics simulations indicate that the adsorption of NAD onto CNTs is driven by interactions between the aromaphilic groups of NAD and the CNT surfaces, leading to coenzyme corona formation. Generally, in living biological systems, the balance of NAD redox (NADH/NAD+ redox) is maintained to sustain metabolism. The present results suggest that CNTs affect coenzyme-dependent metabolic reactions by disrupting the redox balance through coenzyme corona formation and subsequent coenzyme oxidation. The proposed molecular mechanism not only advances the fundamental understanding of the biological impact of CNTs in terms of metabolism but also contributes to biological CNT applications.
Asunto(s)
Coenzimas , Nanotubos de Carbono , Coenzimas/metabolismo , NAD/metabolismo , Oxidación-Reducción , Transporte de ElectrónRESUMEN
Structural proteins such as spider silk and silkworm silk are generally poorly soluble in aqueous and organic solutions, making them difficult to manipulate in manufacturing processes. Although some organic acids and alcohols, such as formic acid and hexafluoroisopropanol (HFIP), effectively solubilize poorly soluble proteins, little is known about their protein solubilization mechanism. In this study, the solubility of N-acetyl amino acid amide compounds in organic solvents-formic acid, acetic acid, HFIP and isopropanol-was measured to clarify the protein solubilization mechanism at the amino acid residue level. On the basis of thermodynamic analyses of the solubility in terms of the transfer free energy (from water to organic solvents), every organic solvent was found to be effective in thermodynamically stabilizing hydrophobic amino acid side chains in the liquid phase. Formic acid and HFIP were comparably effective in the stabilization of the polypeptide backbone, whereas acetic acid and isopropanol were ineffective. Therefore, the significant solubilizing effect of formic acid and HFIP on the structural proteins was attributed to their favorable interactions with hydrophobic amino acid side chains and with the polypeptide backbone of the proteins. The present findings are useful for the optimization of protein manipulation and amino acid sequence design.
Asunto(s)
2-Propanol/química , Amidas/química , Aminoácidos/química , Formiatos/química , Proteínas/química , Solubilidad , Solventes/químicaRESUMEN
Zirconia modified with ethylenediaminetetra(methylenephosphonic acid) (EDTMP) has an affinity for antibodies, including immunoglobulin G (IgG) and immunoglobulin M (IgM). However, little is known about the mechanism underlying antibody selectivity. In this study, we examined the interactions of EDTMP-modified zirconia with proteinogenic amino acids using chromatographic and batch methods to gain mechanistic insights into antibody selectivity at the amino acid level. We demonstrated that EDTMP-modified zirconia has an affinity for amino acids with a positively charged side chain, especially lysine. Similar trends were observed for oligopeptides. This affinity was reduced by the addition of sodium phosphate or sodium polyphosphates. Thus, the antibody selectivity of EDTMP-modified zirconia is primarily ascribable to electrostatic attractions between the EDTMP moieties of the zirconia surfaces and the constant region of antibodies that are rich in lysine residues. Consistent with this, the human IgG antibody has a higher adsorption ability onto EDTMP-modified zirconia than the rabbit IgG antibody, which has fewer lysine residues in the constant region. These findings are useful not only for improving antibody purification but also for developing new applications, including purification of proteins tagged with positively charged amino acid residues.
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Aminoácidos , Circonio , Adsorción , QuelantesRESUMEN
In drug development, water-miscible solvents are commonly used to dissolve drug substances. Typical routine procedures in drug development include dilution of the stock drug solution into an aqueous solution containing target macromolecules for drug binding assays. However, water-miscible solvents impose some technical limitations on the assays on account of their light absorption and heat capacity. Here, we examined the effects of the dilution of 3 water-miscible solvents, that is, dimethyl sulfoxide, acetonitrile, and 1,4-dioxane, on the baseline stability and signal/noise ratio in circular dichroism spectroscopy, isothermal titration calorimetry, and differential scanning calorimetry. Dimethyl sulfoxide and 1,4-dioxane affect the signal/noise ratio of circular dichroism spectra at typically used concentrations due to their light absorbance. The water-miscible solvents generate interfering signals in the isothermal titration calorimetry due to their mixing heat. They show negative or positive slope in the differential scanning calorimetry. Such interfering effects of the solvents are reduced by appropriate dilution according to the analytical techniques. Because the water-miscible solvents have solubilization capacity for alkyl chain moieties and aromatic moieties of chemicals, drug substances containing these moieties can be dissolved into the solvents and then subjected to the analyses to examine their interactions with target proteins after appropriate dilution of the drug solutions.
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Acetonitrilos/química , Aminoácidos/química , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Dimetilsulfóxido/química , Dioxanos/química , Ácido Gálico/química , Solventes/química , Agua/química , Composición de Medicamentos , Ácido Gálico/análogos & derivados , SolubilidadRESUMEN
Nanomaterials introduced into biological systems are immediately coated by proteins in vivo. They induce oxidative stress on adsorbed proteins and hence alter the protein structures, which determines the fate pathways and biological impacts of nanomaterials. Carbon nanotubes (CNTs) have been suggested to cause protein oxidation. In this work, we discovered that CNTs induce oxidative stress on proteins in cooperation with coexisting metals originating from catalyst remains. Protein sulfhydryl groups were readily oxidized by the coexistence of CNTs and metals. Numerical simulations of the reaction demonstrated that the metals effectively mediate electron transfer between the CNTs and protein sulfhydryl groups. Thus, the coexistence of CNTs and metals, even in low concentrations, generates oxidative stress on proteins with high reaction rates. Metal catalysts used for CNT growth, in turn, catalyze the oxidation reaction of proteins. The proposed protein oxidation mechanism will advance the fundamental understanding of the biological safety and toxicity of nanomaterials synthesized using metal catalysts.
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Nanotubos de Carbono/química , Proteínas/química , Elementos de Transición/química , Catálisis , Estrés OxidativoRESUMEN
The adsorption of proteins onto nanoparticles such as carbon nanotubes (CNTs) governs the early stages of nanoparticle uptake into biological systems. Previous studies regarding these adsorption processes have primarily focused on the physical interactions between proteins and nanoparticles. In this study, using reduced lysozyme and intact human serum albumin in aqueous solutions, we demonstrated that CNTs interact chemically with proteins. The CNTs induce the oxidation of cysteine residues of the proteins, which is accounted for by charge transfer from the sulfhydryl groups of the cysteine residues to the CNTs. The redox reaction simultaneously suppresses the intermolecular association of proteins via disulfide bonds. These results suggest that CNTs can affect the folding and oxidation degree of proteins in biological systems such as blood and cytosol.
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Cisteína/química , Nanotubos de Carbono/química , Proteínas/química , Adsorción , Oxidación-ReducciónRESUMEN
The dispersibility and liquid crystal formation of a self-assembled lipid nanotube (LNT) was investigated in a variety of aqueous solutions. As the lipid component, we chose a bipolar lipid with glucose and tetraglycine headgroups, which self-assembled into an LNT with a small outer diameter of 16 to 17 nm and a high axial ratio of more than 310. The LNT gave a stable colloidal dispersion in its dilute solutions and showed spontaneous liquid crystal (LC) alignment at relatively low concentrations and in a pH region including neutral pH. The LNT samples with shorter length distributions were prepared by sonication, and the relationship between the LNT axial ratio and the minimum LC formation concentration was examined. The robustness of the LNT made the liquid crystal stable in mixed solvents of water/ethanol, water/acetone, and water/tetrahydrofuran (1:1 by volume) and at a temperature of up to 90 °C in water. The observed colloidal behavior of the LNT was compared to those of similar 1D nanostructures such as a phospholipid tubule.
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Ácidos Eicosanoicos/química , Glucosa/química , Cristales Líquidos/química , Nanotubos/química , Oligopéptidos/química , Coloides , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cristales Líquidos/ultraestructura , Soluciones , Solventes/química , Sonicación , Temperatura , Agua/químicaRESUMEN
Amphiphilic lipid-poly(ethylene glycol) (LPEG) is widely used for the noncovalent functionalization of graphene nanomaterials (GNMs) to improve their dispersion in aqueous solutions for biomedical applications. However, not much is known about the detachment of LPEGs from GNMs and macrophage uptake of dispersed GNMs in relation to the alkyl chain coverage, the PEG coverage, and the linker group in LPEGs. In this study we examined these relationships using single walled carbon nanohorns (SWCNHs). The high coverage of PEG rather than that of alkyl chains was dominant in suppressing the detachment of LPEGs from SWCNHs in protein-containing physiological solution. Correspondingly, the quantity of LPEG-covered SWCNHs (LPEG-SWCNHs) taken up by macrophages decreased at a high PEG coverage. Our study also demonstrated an effect of the ionic group in LPEG on SWCNH uptake into macrophages. A phosphate anionic group in the LPEG induced lower alkyl chain coverage and easy detachment of the LPEG, however, the negative surface charge of LPEG-SWCNHs reduced the uptake of SWCNHs by macrophages.
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Grafito/química , Lípidos/química , Macrófagos/metabolismo , Nanotecnología , Polietilenglicoles/química , Animales , Ratones , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Polietilenglicoles/metabolismoRESUMEN
A method to control the release of the anti-cancer drug doxorubicin (Dox) from cylindrical nanocapsules, known as organic nanotubes (ONTs), is reported. Co-assembly of a tube-forming glycolipid and its hydrophobized analogue yield novel ONTs with both -COOH and hydrophobic benzyloxycarbonyl groups localized on cylindrical nanochannels. The hydrophobicity of the ONTs nanochannels is easily tunable by adjusting the mixing ratio of the two glycolipids in the co-assembly process. The resultant biologically stable ONTs are able to capture Dox with high efficiency into the cylindrical nanochannels via ion complexation between cationic Dox and anionic -COO(-) , and the release of Dox from hybrid ONTs is effectively controlled by tuning the electrostatic interaction and the hydrophobicity. This controlled release by tuning the hydrophobicity of the ONTs' nanochannels greatly reduces the cytotoxicity of Dox@ONTs for HeLa cells.
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Preparaciones de Acción Retardada/síntesis química , Doxorrubicina/administración & dosificación , Nanocápsulas/química , Nanocápsulas/ultraestructura , Compuestos Orgánicos/química , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Difusión , Doxorrubicina/química , Células HeLa , Humanos , PorosidadRESUMEN
A bipolar glycolipid self-assembles into organic nanotubes upon its chelation with an anticancer drug cis-dichlorodiamineplatinum(II) (CDDP). The facile synthesis of glycolipid, chelation-assisted formation of the nanotubes, and efficient loading and prolonged release of CDDP demonstrate a new approach to high-axial supramolecular drug nanocarriers.
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Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Preparaciones de Acción Retardada/química , Glucolípidos/química , Nanotubos/química , Modelos Moleculares , Nanotubos/ultraestructuraRESUMEN
Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1µm in length was rarely taken up into the cells, while those with a length of 400-800nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.
Asunto(s)
Arginina/química , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Glucolípidos/química , Nanotubos/química , ADN/administración & dosificación , ADN/genética , Portadores de Fármacos/síntesis química , Citometría de Flujo , Glucolípidos/síntesis química , Proteínas Fluorescentes Verdes/genética , Humanos , Células KB , Microscopía Confocal , Microscopía Electrónica de Transmisión de Rastreo , Plásmidos , Propiedades de Superficie , TransgenesRESUMEN
We have synthesized bicyclic azobenzene dimers that possess enantiomers whose racemization rates could be controlled reversibly through E-Z photoisomerization of the azobenzene units. Upon alternating the exposure to r- and l-CPL, we were able to repeatedly perform partial enrichment of (S)- and (R)- enantiomers, respectively.