Interaction-induced structural transformation of lysozyme and kappa-carrageenan in binary complexes.

The interactions between κ-carrageenan and hen egg-white lysozyme have been studied. In dilute solutions, the insoluble complexes with constant κ-carrageenan/lysozyme ratio of 0.3, or 12 disaccharide units per mole of protein are formed. FTIR-spectroscopy revealed that κ-carrageenan retains its unordered conformation and induces the rise of ß-structure in lysozyme. In the complexes formed in concentrated mixtures, κ-carrageenan adopts helical conformation and lysozyme retains its native-like structure. These complexes contain 21 disaccharide units per mole of protein. Molecular modeling showed that flexible coil and rigid double helix of κ-carrageenan have different binding patterns to lysozyme surface. The latter has a strong preference to positively charged spots in lysozyme α-domain while the former also interacts to protein ß-domain and stabilizes short-living ß-structures. The obtained results confirm the preference of unordered κ-carrageenan to ß-structure rich protein regions, which can be further used in the development of carrageenan-based protection of amyloid-like aggregation of proteins.

Spectroscopic, zeta potential and molecular dynamics studies of the interaction of antimicrobial peptides with model bacterial membrane.

Peptide-membrane interactions play a key role in the mechanisms of activity of antimicrobial peptides. Here, methods of fluorescence spectroscopy, zeta potential, and molecular dynamics modeling were used to study the interaction of new antimicrobial peptide megin with model bacterial membrane. The Gibbs free energy of -6 kcal/mol characterizes the interaction of the peptides with liposomes containing DOPE and POPG lipids. Fluorescence data, acrylamide quenching, and MD simulations show that megin peptides are mainly located at the lipid/water interface and are aligned parallel to the bilayer surface in a carpet like manner. Measurements of zeta potential demonstrate the decrease of the negative potential of liposomes in the presence of peptides. The influence of megin on the membrane properties is also confirmed by molecular dynamics simulations. Insertion of peptides into the membrane disturbs lipid ordering, decreases the order parameters of lipids, and facilitates penetration of water molecules through the membrane. According to our results, we proposed that the megin antimicrobial activity can be explained by the carpet model of peptide activity.

Interplay between secondary structure and ion binding upon thermoreversible gelation of κ-carrageenan.

It is widely accepted that cation binding specifically favors chain ordering and gelling of κ-carrageenan. However, current insights into the exact sequence of binding and conformational rearrangements as well as into the structure of binding sites are controversial. In the present work, the FTIR-spectroscopy combined with the computer modelling has been used to reveal the relation between cation binding and the secondary structure transition upon thermoreversible gelation of the κ-carrageenan. Three states of sulfate groups were defined spectroscopically: one cation-free and two specific cation-bound states. The DFT calculations reveal two energetically inequivalent spatial structures of cation binding unit, formed due to the local conformational adjustment in neocarrabiose moiety. Besides a charge screening effect, the cation-bound conformation of neocarrabiose also favors the helix formation.

Molecular structure and properties of κ-carrageenan-gelatin gels.

Rheological studies, FTIR spectroscopy and a molecular docking approach were used to explore the structural basis of the peculiar physicochemical properties of gelatin gels modified with a κ-carrageenan admixture. Mixed gel properties are affected by the polysaccharide-to-gelatin ratio, Z, and can be divided into two categories. At low ratios, the strength of mixed gels varies insignificantly compared to gelatin due to the similar structures of the gels. Above the threshold content of κ-carrageenan (Z > 0.1), the storage modulus and yield stress of mixed gels are significantly enhanced. The nonadditivity and threshold character of the rheological properties could be the result of conformational ordering of both gelatin and κ-carrageenan, leading to the formation of additional junction zones in the gel network. According to molecular docking studies, the junctions could be formed as a result of complementary interactions between the gelatin triple helix and the κ-carrageenan double helix. The stack formation increases the interaction energy, which explains the strengthening of the gel network.

Effect of Lipid Surface Composition on the Formation and Structure of Fibrin Clots.

We studied the influence of lipid surface composition on the kinetics of fibrin clot formation and its structure. It was shown that lipid surface affects all phases of fibrin polymerization and chances clot morphology. The magnitude and character of the effect depend on the charge and phase state of lipids that determine the interaction of fibrinogen with the lipid surface and its conformational changes, which modulated the process of fibrinogen conversion into fibrin and, as a result, the formation and morphology of the fibrin clot.

[Structure and Activity of Fungal Lipases in Bile Salt Solutions].

The changes in structure and catalytic properties of fungal lipases (Candida rugosa, Rhizomucor miehei, Mucor javanicus) were investigated in micellar solutions of bile salts that differ in hydrophilic-lypophilic balance and reaction medium properties. The methods of circular dichroism and tryptophan fluorescence were applied to estimate the changes in peptide structure within complexes with bile salt micelles. Bile salts do not exert a significant influence on the structure of the enzymes under study: in Rh. miehei and M. javanicus lipases the alpha helix content slightly decreased, the influence of bile salts on the C. rugosa structure was not revealed. Despite negligible structural modifications in the enzymes, in bile salt solutions a considerable change in their catalytic properties was observed: an abrupt decrease in catalytic effectiveness. Substrate-bile salts micelles complex formation was demonstrated by the NMR self-diffusion method. The model of a regulation of fungal lipase activity was proposed.

Tissue-specific rhamnogalacturonan I forms the gel with hyperelastic properties.

Rhamnogalacturonans I are complex pectin polysaccharides extremely variable in structure and properties and widely represented in various sources. The complexity and diversity of the structure of rhamnogalacturonans I are the reasons for the limited information about the properties and supramolecular organization of these polysaccharides, including the relationship between these parameters and the functions of rhamnogalacturonans I in plant cells. In the present work, on the example of rhamnogalacturonan I from flax gelatinous fibers, the ability of this type of pectic polysaccharides to form at physiological concentrations hydrogels with hyperelastic properties was revealed for the first time. According to IR spectroscopy, water molecules are more tightly retained in the gelling rhamnogalacturonan I from flax fiber cell wall in comparison with the non-gelling rhamnogalacturonan I from primary cell wall of potato. With increase in strength of water binding by rhamnogalacturonan I, there is an increase in elastic modulus and decrease in Poisson's ratio of gel formed by this polysaccharide. The model of hyperelastic rhamnogalacturonan I capture by laterally interacting cellulose microfibrils, constructed using the finite element method, confirmed the suitability of rhamnogalacturonan I gel with the established properties for the function in the gelatinous cell wall, allowing consideration of this tissue- and stage-specific pectic polysaccharide as an important factor in creation of gelatinous fiber contractility.

Lipoplexes of dicationic gemini surfactants with DNA: Structural features of DNA compaction and transfection efficiency.

The internal structure of DNA lipoplexes with hydroxyethylated alkylammonium gemini surfactants (GS) with high transfection activity was studied by circular dichroism. It was shown that the efficiency of transfection of HEK293T cells with the pEGFP-N1 circular plasmid was different from zero only in the region of existence of chiral supramolecular DNA-GS complexes and reaches a maximum at concentrations at which the spontaneous aggregation of components is observed.

[Self-association and secondary structure of beta-casein].

The secondary structure alterations, accompanying isothermal and temperature guided beta-casein micellization have been studied by dynamic light scattering, circular dichroism and Fourier transform infrared spectroscopy techniques. Micelle formation induced by increase of protein concentration at constant temperature is accompanied by the formation of scanty number of additional peptide hydrogen bonds, preliminary assigned to intraprotein beta-structure. Heating results in more pronounced but qualitatively different changes consisted in dehydration of peptide groups and disruption of polyproline II helix segments with subsequent conversion to random and beta-turns. Nevertheless, in both cases the total number of residues involved in transition is quite few and cannot be regarded as a decisive factor for casein micellization.

[Influence of dioxane on the hydration shell of polypeptides].

Our interest in modifying influence of the low-molecular organic compounds on the hydration shell of biopolymers is due to a well-known fact that the former can act as regulators of the enzymatic activity changed the hydration shell of the latter. Dioxane was chosen because of its wide application in non-aqueous biocatalysis. In the present study we investigate the mechanisms of the dioxane influence on water of the first hydration layer of the model polypeptides by FTIR-spectroscopy during simultaneous sorption of water-dioxane vapors into the polypeptide films at low water activity. It was found that modification of the hydration shell of the studied polypeptides was mainly due to the indirect ordering ofpolypeptide secondary structure at penetration of dioxane molecules at these conditions.

[Chaperone-like activity of beta-casein and thermal stability of alcohol dehydrogenase].

To elucidate the correlation of structural peculiarities of beta-casein and their chaperon-like activity the modified forms of the protein (with cysteinyl residues introduced in polypeptide chain) were investigated. The aggregation of native and recombinant beta-caseins was studied as well as their chaperon-like activity towards alcohol dehydrogenase thermal aggregation. It was shown that physico-chemical and chaperone-like properties ofdimeric and oligomeric forms ofbeta-casein (which formation is due to intermolecular disulfide bonds) differ significantly from monomeric forms. It was found that thermal stability of alcohol dehydrogenase depends on beta-casein concentration.

Ethylene glycol and the thermostability of trypsin in a reverse micelle system.

The influence of ethylene glycol (EG) on the kinetics of hydrolysis of N-alpha-benzoyl-L-arginine ethyl ether catalyzed by trypsin encapsulated in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT)-based reverse micelles was studied at different temperatures. Ethylene glycol was shown to shift the range of the trypsin activity in the reverse micelles towards higher temperatures. Infrared spectroscopy showed a stabilizing effect of EG on the secondary structure of the protein in the system of reverse micelles. Electron spin resonance spectroscopy showed that the solubilized protein affected the interactions of EG with the polar head groups of AOT and altered the rigidity of the micellar matrix. The results indicate that EG increases the thermostability of the solubilized enzyme in microemulsion media by two mechanisms.

[The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. I. Influence pH, temperature and ligand binding].

The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.

FTIR-spectroscopic studies of the fine structure of nitrocellulose treated by Desulfovibrio desulfuricans.

Most studies have concluded that nitrocellulose (NC) with high degree of nitrogen content is resistant to biodegradation. Our results demonstrated that NC (>11%N) does undergo biotransformation in the presence of sulfate-reducing bacteria Desulfovibrio desulfuricans 1388. FTIR analyses indicated that the substitution of nitro groups for OH(-) groups took place. The spectrum of precipitate obtained after acetone extraction of NC resembled mainly the spectrum of native cellulose. Thus the synthetic unbiodegradable polymer was transformed to the natural compound accessible for microorganisms.

Calorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents.

Calorimetric heat effects and structural rearrangements assessed by means of Fourier transform infrared (FTIR) amide I spectra were followed by immersing dry human serum albumin and bovine pancreatic alpha-chymotrypsin in low water organic solvents and in pure water at 298 K. Enthalpy changes upon immersion of the proteins in different media are in a good linear correlation with the corresponding IR absorbance changes. Based on calorimetric and FTIR data the solvents were divided into two groups. The first group includes carbon tetrachloride, benzene, nitromethane, acetonitrile, 1,4-dioxane, n-butanol, n-propanol and pyridine where no significant heat evolution and structural changes were found during protein immersion. Due to kinetic reasons no significant protein-solvent interactions are expected in such systems. The second group of solvents includes dimethyl sulfoxide, methanol, ethanol, and water. Immersion of proteins in these media results in protein swelling and involves significant exothermic heat evolution and structural changes in the protein. Dividing of different media in the two groups is in a qualitative correlation with the solvent hydrophilicity defined as partial excess molar Gibbs free energy of water at infinite dilution in a given solvent. The first group includes the solvents with hydrophilicity exceeding 2.7 kJ/mol. More hydrophilic second group solvents have this energy values less than 2.3 kJ/mol. The hydrogen bond donating ability of the solvents also assists in protein swelling. Hydrogen bonding between protein and solvent is assumed to be a main factor controlling the swelling of dry solid proteins in the studied solvents.