RESUMEN
This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme's active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology.
Asunto(s)
Bromelaínas , Papaína , Papaína/metabolismo , Ficaína/química , Ficaína/metabolismo , Carboximetilcelulosa de Sodio , Simulación del Acoplamiento Molecular , Polímeros , Cloruro de Sodio , Cloruro de Sodio Dietético , SodioRESUMEN
Herein, we aimed to highlight current "gaps" in the understanding of the potential interactions between the Anle138b isomer ligand, a promising agent for clinical research, and the intrinsically disordered alpha-synuclein protein. The presence of extensive unstructured areas in alpha-synuclein determines its existence in the cell of partner proteins, including the cyclophilin A chaperone, which prevents the aggregation of alpha-synuclein molecules that are destructive to cell life. Using flexible and cascaded molecular docking techniques, we aimed to expand our understanding of the molecular architecture of the protein complex between alpha-synuclein, cyclophilin A and the Anle138b isomer ligand. We demonstrated the possibility of intricate complex formation under cellular conditions and revealed that the main interactions that stabilize the complex are hydrophobic and involve hydrogen.
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Ciclofilina A , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Simulación del Acoplamiento Molecular , Ligandos , Amiloide/metabolismo , Proteínas AmiloidogénicasRESUMEN
Rheumatoid arthritis belongs to the group of chronic systemic autoimmune diseases characterized by the development of destructive synovitis and extra-articular manifestations. Cytokines regulate a wide range of inflammatory processes involved in the pathogenesis of rheumatoid arthritis and contribute to the induction of autoimmunity and chronic inflammation. Janus-associated kinase (JAK) and signal transducer and activator of transcription (STAT) proteins mediate cell signaling from cytokine receptors, and are involved in the pathogenesis of autoimmune and inflammatory diseases. Targeted small-molecule drugs that inhibit the functional activity of JAK proteins are used in clinical practice for the treatment of rheumatoid arthritis. In our study, we modeled the interactions of the small-molecule drug ruxolitinib with JAK1 and JAK2 isoforms and determined the binding selectivity using molecular docking. Molecular modeling data show that ruxolitinib selectively binds the JAK1 and JAK2 isoforms with a binding affinity of -8.3 and -8.0 kcal/mol, respectively. The stabilization of ligands in the cavity of kinases occurs primarily through hydrophobic interactions. The amino acid residues of the protein globules of kinases that are responsible for the correct positioning of the drug ruxolitinib and its retention have been determined.
Asunto(s)
Artritis Reumatoide , Janus Quinasa 2 , Aminoácidos , Artritis Reumatoide/tratamiento farmacológico , Citocinas , Humanos , Janus Quinasa 1 , Janus Quinasa 2/metabolismo , Quinasas Janus , Simulación del Acoplamiento Molecular , Nitrilos , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles , Pirimidinas , Receptores de CitocinasRESUMEN
Phospholipase A of the bacterial outer membrane (OMPLA) is a ß-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2--palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)--with OMPLA and considers a possible involvement of the enzyme in the Ca²âº-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca²âº-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²âº and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.
Asunto(s)
Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Fosfolipasas A2/química , Fosfolipasas A/química , Ácidos Aristolóquicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Cetonas/farmacología , Modelos Moleculares , Conformación Molecular , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas A2/metabolismo , Conformación Proteica , Transformación Bacteriana/efectos de los fármacosRESUMEN
The temperature dependence of the partial heat capacity of the native protein structure in an aqueous solution has been analyzed. It is shown that the strictly linear temperature dependence is due to the contributions of the vibrational and conformational components, which indicates volume consistensy and the absence of conformational transitions up to the main two-state transition. The two-level structural and functional organization of the protein three-dimensional structure are considered in relation to the energy and conformational entropy properties in accordance with the principles of the organization of the protein macromolecule.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Calor , Proteínas , Termodinámica , Proteínas/química , Entropía , Temperatura , Desnaturalización ProteicaRESUMEN
The present work is devoted to research on the interaction between carboxymethyl cellulose sodium salt and its derivatives (graft copolymer of carboxymethyl cellulose sodium salt and N,N-dimethyl aminoethyl methacrylate) with cysteine protease (ficin). The interaction was studied by FTIR and by flexible molecular docking, which have shown the conjugates' formation with both matrices. The proteolytic activity assay performed with azocasein demonstrated that the specific activities of all immobilized ficin samples are higher in comparison with those of the native enzyme. This is due to the modulation of the conformation of ficin globule and of the enzyme active site by weak physical interactions involving catalytically valuable amino acids. The results obtained can extend the practical use of ficin in biomedicine and biotechnology.
RESUMEN
Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.
RESUMEN
Although amino acids are insensitive to visible light, as is generally accepted, we show that particular light-matter interaction can break this obviousness. Using sensitive (FT-IR)-technique in a combination of a broadband visible light source, we registered emission spectra of glycine in the range 2500-500 cm-1. Sensitivity of the infrared emission spectrum to the exciting power -induced changes in the glycine structure was demonstrated experimentally.Vibrational spectra of glycine displayed the prominent spectral features of CH2, COO-, COOH, NH+3 groups in the "fingerprint region". Simultaneous appearance of ionised COO- and unionised COOH forms of glycine in solution at neutral pH clearly indicated that visible light induces the partial protonation of COO- groups; if so, visible light irradiation should lead to occurrence of dimers or dimeric hydrogen - bonded structures. Spectroscopic and microscopic evidence of visible light-mediated formation of aggregates and nucleus in aqueous solution was presented.Electronic absorption/emission spectra of glycine in aqueous solution were primarily characterized in the near ultraviolet-visible region (240-600 nm). Negligible absorption near 270 nm was observed for a 1.0 M solution and dramatically enhanced with its "aging". Moreover, an extension of the absorption edge into the region above 400 nm could be seen. Due to the visible light irradiation, we observed modification of electronic structure or occurrence of additional species causing changes in absorption of glycine amino acid. For "aged" solution, it was shown that excitation spectra corresponding to the different emission wavelengths were entirely different, at that each excitation-spectral band had a characteristic emission band.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Glicina , Análisis de Fourier , Concentración de Iones de Hidrógeno , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).
Asunto(s)
Bromelaínas/química , Bromelaínas/metabolismo , Quitosano/metabolismo , Composición de Medicamentos/métodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Ficaína/química , Ficaína/metabolismo , Papaína/química , Papaína/metabolismo , Adsorción , Ananas/enzimología , Biocatálisis , Biotecnología/métodos , Carica/enzimología , Dominio Catalítico , Estabilidad de Enzimas , Ficus/enzimología , Concentración de Iones de Hidrógeno , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Estructura Secundaria de Proteína , TemperaturaRESUMEN
Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain â ficin â papain. After the UV irradiation with 151â¯J·m-2 intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151-6040â¯J·m-2 range of irradiation intensities. A decrease in the catalytic capacity of ficin and the increase of its globule size occurred after exposure to a radiation of 3020â¯J·m-2 intensity. The decrease in papain activity was observed at the UV irradiation intensity of 453â¯J·m-2, and an increase of the papain globule size was detected at 755â¯J·m-2. Immobilization on chitosan matrix leads to the increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be postulated that the chitosan matrix acts as photoprotector for immobilized ficin, bromelain and papain. The obtained results can be helpful for development of drugs based on chitosan and cysteine proteases in combination with phototherapy, as well as for choosing their sterilization conditions.
Asunto(s)
Bromelaínas/metabolismo , Ficaína/metabolismo , Papaína/metabolismo , Rayos Ultravioleta , Biocatálisis/efectos de la radiación , Bromelaínas/química , Quitosano/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Ficaína/química , Cinética , Papaína/química , Estructura Terciaria de ProteínaRESUMEN
We develop a technique for the sorption of inulinase from Kluyveromyces marxianus on the KU-2 matrix cation-exchanger. The most appropriate conditions for immobilization are: 25⯰C, pHâ¯4.5, incubation time 1.5â¯h, protein content during immobilization 10â¯mg/g of carrier. At higher (20-100â¯mg/g) concentrations, inulinase forms local high-concentration domains on the ion-exchanger surface, the enzyme is adsorbed on the protein, not on the carrier. 62% of the initial catalytic activity of inulinase is immobilized on KU-2 by the adsorption method. Upon the enzyme immobilization on KU-2, the number of unordered structures in the protein is reduced by ~10%, that points to the compaction of the molecule. Hydrogen bonds are formed only between the sulfo groups of carrier and the protein molecule, without affecting other structures of the cation exchanger. The highest activity of the inulinase immobilized on KU-2 was observed at 70⯰C (cf. 50⯰C for the native enzyme). Heating of the solution for 60â¯min in the temperature range of 50-80⯰C failed to inactivate completely the immobilized enzyme; the complete loss of its ability to hydrolyze inulin was achieved only after more than 1â¯h incubation at 90⯰C.
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Enzimas Inmovilizadas , Glicósido Hidrolasas/química , Resinas de Intercambio Iónico/química , Adsorción , Biocatálisis , Activación Enzimática , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Kluyveromyces/enzimología , Modelos Moleculares , Conformación Proteica , Análisis Espectral , TemperaturaRESUMEN
The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the 'gas phase'. It was shown that specific values of the energy of weak intramolecular bonds (∆Hint), conformational free energy (∆Gconf) and entropy (∆Sconf) are the same for proteins with molecular weight 7-25 kDa. Using the energy value (∆Hint) and the proposed approach for estimation of the conformational entropy of native protein (SNC), numerical values of the absolute free energy (GNC) were obtained.
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Conformación Proteica , Pliegue de Proteína , Proteínas/química , Termodinámica , Entropía , Temperatura , Agua/químicaRESUMEN
We performed thermodynamic analysis of temperature-induced unfolding of mesophilic and thermophilic proteins. It was shown that the variability in protein thermostability associated with pH-dependent unfolding or linked to the substitution of amino acid residues on the protein surface is evidence of the governing role of the entropy factor. Numerical values of conformational components in enthalpy, entropy and free energy which characterize protein unfolding in the "gas phase" were obtained. Based on the calculated absolute values of entropy and free energy, a model of protein unfolding is proposed in which the driving force is the conformational entropy of native protein, as an energy of the heat motion (T·S(NC)) increasing with temperature and acting as an factor devaluating the energy of intramolecular weak bonds in the transition state.
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Desnaturalización Proteica , Pliegue de Proteína , Proteínas/química , Entropía , Calor , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , TermodinámicaRESUMEN
We propose a hypothesis that the T-cell receptor is a possible target of thymic hormones. We modelled the conformational dynamics of thymopentin and its structural variants in solution, as well as the interactions of these short peptides with the proposed molecular target. Thymopentin is a five-amino-acid fragment of the thymic hormone thymopoietin (residues 32 to 36) that reproduces the immunomodulatory activity of the complete hormone. Using molecular dynamics and flexible docking methods, we demonstrated high-affinity binding of thymopentin and its prospective mimetics with the T-cell receptor. The calculated biological activity spectra of thymopentin and its two promising modifications can be used in immunomodulatory activity screenings with live systems.