Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-ß structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-ß structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like ß-structures in HEWL, retaining most of the cross-ß structures. In contrast, λ-carrageenan even increased the content of amyloid cross-ß structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-ß structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

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.

Protein Translational Diffusion and Intermolecular Interactions of Globular and Intrinsically Unstructured Proteins.

The study of intermolecular interactions of proteins has been an important problem for many years. This paper presents an approach to analyze different levels of protein interactions in solutions through a set of the second- and higher-order virial coefficients. The proposed approach is based on the diversified analysis of protein translational collective diffusion and self-diffusion obtained by dynamic light scattering and the pulsed-field gradient NMR (PFG NMR) spectroscopy experimental data. The experimental results were analyzed within the theoretical approach based on Vink's frictional formalism of nonequilibrium thermodynamics and the standard Derjaguin-Landau-Verwey-Overbeekb (DLVO) theory of interactions of colloid particles in electrolyte solutions. The second- and higher-order virial coefficients were obtained to estimate the pairwise and many-body intermolecular interactions in the solutions of globular α-chymotrypsin and intrinsically unstructured αS-casein. The second virial coefficients were calculated from the model of the protein-protein potential of mean force. The description of protein-protein interactions includes a set of interaction potentials: the attractive charge-dipole, dipole-dipole, the dispersion Hamaker, the mean force osmotic-attraction, and the repulsive charge-charge ones. It has been found that the major contribution to the intermolecular αS-casein interactions is made by the repulsive charge-charge potential, whereas for the case of α-chymotrypsin, the contributions from other types of interaction are of importance. It was determined that the model was well suited to describe the interactions of both globular and intrinsically disordered proteins. The suggested combination of Vink's approach and the DLVO theory is novel and holds much promise to make a profound analysis of the processes in systems containing various types of protein molecules.

Rheological Properties of Fish and Mammalian Gelatin Hydrogels as Bases for Potential Practical Formulations.

Hydrogels have the ability to retain large amounts of water within their three-dimensional polymer matrices. These attractive materials are used in medicine and the food industry; they can serve as the basis for structured food products, additives, and various ingredients. Gelatin is one of widely used biopolymers to create hydrogels that exhibit biocompatibility and tunable rheological properties. In this study, we offer a comparative analysis of rheological properties of gelatin-based hydrogels (C = 6.67%), including mammalian gelatins from bovine and porcine skins and fish gelatins from commercial samples and samples extracted from Atlantic cod skin. Mammalian gelatins provide high strength and elasticity to hydrogels. Their melting point lies in the range from 22 to 34 °C. Fish gelatin from cod skin also provides a high strength to hydrogels. Commercial fish gelatin forms weak gels exhibiting low viscoelastic properties and strength, as well as low thermal stability with a melting point of 7 °C. Gelatins were characterized basing on the analysis of amino acid composition, molecular weight distribution, and biopolymer secondary structure in gels. Our research provides a unique rheological comparison of mammalian and fish gelatin hydrogels as a tool for the re-evaluation of fish skin gelatin produced through circular processes.

Supramolecular design of biocompatible nanocontainers based on amphiphilic derivatives of a natural compound isosteviol.

Two diterpenoid surfactants with ammonium head groups and bromide (S1) or tosylate (S2) counterions have been synthesized. Exploration of these biomimetic species made it possible to demonstrate that even minor structural changes beyond their chemical nature may dramatically affect their solution behavior. While their aggregation thresholds differ inconsiderably, morphological behavior and affinity to lipid bilayer are strongly dependent on the counterion nature. Compound S2 demonstrates properties of typical surfactants and forms small micelle-like aggregates above critical micelle concentration. For surfactant S1, two critical concentrations and two types of aggregates occur. Structural transitions have been observed between small micelles and aggregates with higher aggregation numbers and hydrodynamic diameter of ca. 150 nm. Unlike S2, surfactant S1 is shown to integrate with liposomes based on dipalmitoylphosphatidylcholine, resulting in a decrease of the temperature of the main phase transition. Both surfactants demonstrate an effective complexation capacity toward oligonucleotide (ONu), which is supported by recharging the surfactant-ONu complexes and the ethidium bromide exclusion at a low N/P ratio. Meanwhile, a very weak complexation of plasmid DNA with the surfactants has been revealed in the gel electrophoresis experiment. The DNA transfer to bacterial cells mediated by the surfactant S1 is shown to depend on the protocol used. In the case of the electroporation, the inhibition of the cell transformation occurs in the presence of the surfactant, while upon the chemical treatment no surfactant effect has been observed. The variability in the morphology, the biocompatibility, the nanoscale dimension and the high binding capacity toward the DNA decamer make it possible to nominate the designed surfactants as promising carriers for biosubstrates or as a helper surfactant for the mixed liposome-surfactant nanocontainers.

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.

The α-helix to ß-sheet transition in stretched and compressed hydrated fibrin clots.

Fibrin is a protein polymer that forms the viscoelastic scaffold of blood clots and thrombi. Despite the critical importance of fibrin deformability for outcomes of bleeding and thrombosis, the structural origins of the clot's elasticity and plasticity remain largely unknown. However, there is substantial evidence that unfolding of fibrin is an important part of the mechanism. We used Fourier transform infrared spectroscopy to reveal force-induced changes in the secondary structure of hydrated fibrin clots made of human blood plasma in vitro. When extended or compressed, fibrin showed a shift of absorbance intensity mainly in the amide I band (1600-1700 cm(-1)) as well as in the amide II and III bands, indicating an increase of the ß-sheets and a corresponding reduction of the α-helices. The structural conversions correlated directly with the strain or pressure and were partially reversible at the conditions applied. The additional absorbance observed at 1612-1624 cm(-1) was characteristic of the nascent interchain ß-sheets, consistent with protein aggregation and fiber bundling during clot deformation observed using scanning electron microscopy. We conclude that under extension and/or compression an α-helix to ß-sheet conversion of the coiled-coils occurs in the fibrin clot as a part of forced protein unfolding.

Chitosan Graft Copolymers with N-Vinylimidazole as Promising Matrices for Immobilization of Bromelain, Ficin, and Papain.

This work aims to synthesize graft copolymers of chitosan and N-vinylimidazole (VI) with different compositions to be used as matrices for the immobilization of cysteine proteases-bromelain, ficin, and papain. The copolymers are synthesized by free radical solution copolymerization with a potassium persulfate-sodium metabisulfite blend initiator. The copolymers have a relatively high frequency of grafting and yields. All the synthesized graft copolymers are water-soluble, and their solutions are characterized by DLS and laser Doppler microelectrophoresis. The copolymers are self-assembled in aqueous solutions, and they have a cationic nature and pH-sensitivity correlating to the VI content. The FTIR data demonstrate that synthesized graft copolymers conjugate cysteine proteases. The synthesized copolymer adsorbs more enzyme macromolecules compared to non-modified chitosan with the same molecular weight. The proteolytic activity of the immobilized enzymes is increased up to 100% compared to native ones. The immobilized ficin retains up to 97% of the initial activity after a one-day incubation, the immobilized bromelain retains 69% of activity after a 3-day incubation, and the immobilized papain retains 57% of the initial activity after a 7-day incubation. Therefore, the synthesized copolymers can be used as matrices for the immobilization of bromelain, ficin, and papain.

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.

Spatial structures of rhamnogalacturonan I in gel and colloidal solution identified by 1D and 2D-FTIR spectroscopy.

Rhamnogalacturonan I (RG-I), a polysaccharide found in different types of plant cell walls, fulfills specific functions, the structural basis of which remains unclear. Generalized 2D correlation FTIR spectroscopy with dehydration was employed to reveal the structure and interactions in flax RG-I solution and microwave treated gel. Varying water content allowed emphasizing a role of solvent in maintaining different structures. In the gel, 2D correlation maps prove the existence of a conformationally uniform highly hydrated structure. Such a structure is supposed to correspond to non-associated galactan helices stabilized by rare junctions. In colloidal solution the side chains of RG-I associate heterogeneously due to constrains imposed by stiff backbone. Galactan-enriched fraction of RG-I with enzymatically cleaved backbone revealed the tendency of galactan chains to strongly associate in solution. The obtained results shed light on the possible role of backbone and side chains in RG-I spatial organization and confirm the sensitivity and potential of 2D correlation FTIR spectroscopy to probe local ordered structures in non-crystalline polysaccharides.

Theoretical and Experimental Investigation of the Translational Diffusion of Proteins in the Vicinity of Temperature-Induced Unfolding Transition.

Translational diffusion is the most fundamental form of transport in chemical and biological systems. The diffusion coefficient is highly sensitive to changes in the size of the diffusing species; hence, it provides important information on the variety of macromolecular processes, such as self-assembly or folding-unfolding. Here, we investigate the behavior of the diffusion coefficient of a macromolecule in the vicinity of heat-induced transition from folded to unfolded state. We derive the equation that describes the diffusion coefficient of the macromolecule in the vicinity of the transition and use it to fit the experimental data from pulsed-field-gradient nuclear magnetic resonance (PFG NMR) experiments acquired for two globular proteins, lysozyme and RNase A, undergoing temperature-induced unfolding. A very good qualitative agreement between the theoretically derived diffusion coefficient and experimental data is observed.

Structure of Scots pine defensin 1 by spectroscopic methods and computational modeling.

Defensins are part of the innate immune system in plants with activity against a broad range of pathogens, including bacteria, fungi and viruses. Several defensins from conifers, including Scots pine defensin 1 (Pinus sylvestris defensin 1, (PsDef1)) have shown a strong antifungal activity, however structural and physico-chemical properties of the family, needed for establishing the structure-dynamics-function relationships, remain poorly characterized. We use several spectroscopic and computational methods to characterize the structure, dynamics, and oligomeric state of PsDef1. The three-dimensional structure was modeled by comparative modeling using several programs (Geno3D, SWISS-MODEL, I-TASSER, Phyre(2), and FUGUE) and verified by circular dichroism (CD) and infrared (FTIR) spectroscopy. Furthermore, FTIR data indicates that the structure of PsDef1 is highly resistant to high temperatures. NMR diffusion experiments show that defensin exists in solution in the equilibrium between monomers and dimers. Four types of dimers were constructed using the HADDOCK program and compared to the known dimer structures of other plant defensins. Gaussian network model was used to characterize the internal dynamics of PsDef1 in monomer and dimer states. PsDef1 is a typical representative of P. sylvestris defensins and hence the results of this study are applicable to other members of the family.

Structural, biocomplexation and gene delivery properties of hydroxyethylated gemini surfactants with varied spacer length.

Gemini surfactants with hexadecyl tails and hydroxyethylated head groups bridged with tetramethylene (G4), hexamethylene (G6) and dodecamethylene (G12) spacers were shown to self-assemble at the lower critical micelle concentration compared to their conventional m-s-m analogs. The lipoplex formation and the plasmid DNA transfer into different kinds of host cells were studied. In the case of eukaryotic cells, high transfection efficacy has been demonstrated for DNA-gemini complexes, which increased as follows: G6G4>G12 has been obtained in the case of transformation of bacterial cells with plasmid DNA-gemini complexes, mediated by electroporation technique. Solely G6 shows transformation efficacy exceeding the control result (uncomplexed DNA), while the inhibitory effect occurs for G4 and G12. Analysis of physico-chemical features of single surfactants and lipoplexes shows that compaction and condensation effects change as follows: G6

Novel biomimetic systems based on amphiphilic compounds with a diterpenoid fragment: role of counterions in self-assembly.

Novel biomimetic systems are designed based on cationic surfactants composed of an isosteviol moiety and different counterions, namely bromide (S1) and tosylate (S2). The counterion structure is shown to play a crucial role in the surfactant association. A number of methods used provide evidence that only one type of aggregate, i.e., micelles are observed in the S2 systems, while a concentration-dependent association occurs in the case of S1. The DLS and fluorescence anisotropy measurements reveal that the micelle-vesicle-micelle transitions probably occur with the S1 system. The occurrence of small aggregates near the critical micelle concentration with radii of 2.5 nm is supported by NMR self-diffusion data. The Orange OT solubilization results strongly support the idea of a second threshold in the S1 system around 0.025 mM and provide evidence that hydrophobic domains occur in the aggregates. The latter property and the capacity to integrate with the lipid bilayer make it possible to suggest the newly synthesized surfactants as effective nanocontainers for hydrophobic guests.

Interactions of ß-lactoglobulin with serotonin and arachidonyl serotonin.

ß-Lactoglobulin (ß-LG) is a lipocalin, which is the major whey protein of cow's milk and the milk of other mammals. However, it is absent from human milk. The biological function of ß-LG is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. ß-LG has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5-hydroxytryptamine, 5-HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA-5HT), with ß-LG was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with ß-LG forming equimolar complexes. The binding constant for the serotonin/ß-LG interaction is between 105 and 106 M(-1) , whereas for the AA-5HT/ß-LG complex it is between 104 and 105 M(-1) as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25% EtOH). The interactions between serotonin/ß-LG and AA-5HT/ß-LG may compete with self-association (micellization) of both the ligand and the protein. According to far- and near-UV CD results, these ligands have no apparent influence on ß-LG secondary structure, however they partially destabilize its tertiary structure. Their binding by ß-LG may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk-fed animals.