Complexation of Bromelain, Ficin, and Papain with the Graft Copolymer of Carboxymethyl Cellulose Sodium Salt and N-Vinylimidazole Enhances Enzyme Proteolytic Activity.

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.

Carboxymethyl Cellulose-Based Polymers as Promising Matrices for Ficin Immobilization.

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.

Thermodynamics of globular protein native structure.

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.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

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.

In Silico Study of the Interactions of Anle138b Isomer, an Inhibitor of Amyloid Aggregation, with Partner Proteins.

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.

Light-induced effects in glycine aqueous solution studied by Fourier transform infrared-emission spectroscopy and ultraviolet-visible spectroscopy.

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.

Immobilization of inulinase on KU-2 ion-exchange resin matrix.

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, pH 4.5, incubation time 1.5 h, protein content during immobilization 10 mg/g of carrier. At higher (20-100 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 60 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 1 h incubation at 90 °C.

Thermodynamics of globular proteins.

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.

Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eucaryotic phospholipases A2 and their effect on the Ca²âº-induced permeabilization of the bacterial membrane.

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 Ca²âº-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 Ca²âº-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 Ca²âº 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.

Structural and dynamic properties of thymopoietin mimetics.

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.

The entropic nature of protein thermal stabilization.

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.