Marián Antalík

Acridine derivatives inhibit lysozyme aggregation

We have screened a library of structurally distinct acridine derivatives (19 compounds) for their ability to inhibit lysozyme amyloid aggregation in vitro. Studied acridines were divided into three structurally different groups depending on the molecule planarity and type of the side chain—planar acridines, spiroacridines and tetrahydroacridines. Thioflavine T fluorescence assay and transmission electron microscopy were used for monitoring the inhibiting activity of acridines. We have found that both the structure of the acridine side chains and molecule planarity influence their antiamyloidogenic activity. The planar acridines inhibited lysozyme aggregation effectively. Spiroacridines and tetrahydroacridines had no significant effect on the prevention of lysozyme fibrillization, probably resulting from the presence of the heterocyclic 5-membered ring and non-planarity of molecule. Moreover, in the presence of some tetrahydroacridines the enhanced extent of aggregation was detected. We identified the most active acridine derivates from studied compound library characterized by low micromolar IC50 values, which indicate their possible application for therapeutic purpose.

Viscosity and diffusion: crowding and salt effects in protein solutions

We report on a joint experimental–theoretical study of collective diffusion in, and static shear viscosity of solutions of bovine serum albumin (BSA) proteins, focusing on the dependence on protein and salt concentration. Data obtained from dynamic light scattering and rheometric measurements are compared to theoretical calculations based on an analytically treatable spheroid model of BSA with isotropic screened Coulomb plus hard-sphere interactions. The only input to the dynamics calculations is the static structure factor obtained from a consistent theoretical fit to a concentration series of small-angle X-ray scattering (SAXS) data. This fit is based on an integral equation scheme that combines high accuracy with low computational cost. All experimentally probed dynamic and static properties are reproduced theoretically with an at least semi-quantitative accuracy. For lower protein concentration and low salinity, both theory and experiment show a maximum in the reduced viscosity, caused by the electrostatic repulsion of proteins. On employing our theoretical and experimental results, the applicability range of a generalized Stokes–Einstein (GSE) relation connecting viscosity, collective diffusion coefficient, and osmotic compressibility, proposed by Kholodenko and Douglas [Phys. Rev. E, 1995, 51, 1081] is examined. Significant violation of the GSE relation is found, both in experimental data and in theoretical models, in concentrated systems at physiological salinity, and under low-salt conditions for arbitrary protein concentrations.

Effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat‐induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature‐dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

Polyanion Hydrophobicity and Protein Basicity Affect Protein Stability in Protein-Polyanion Complexes

Stability of four dissimilar basic proteins (chymotrypsinogen A, ribonuclease A, cytochrome c, lysozyme) in the complex with four polyanions (heparin, poly(vinylsulfate), poly(4-styrene-sulfonate), Nafion) has been studied by differential scanning calorimetry. The polyanions were chosen because of their different charge density and hydrophobicity. Relative hydrophobicity of polyanions have been compared by three different parameters: (i) partition coefficient determined in octanol/water system, (ii) electrocapillary curves obtained by the method of controlled convection, and (iii) change in absorbance of small cationic amphiphilic molecule, aminoacridine, due to interaction with polyanion. Our results suggest that stability of proteins in the complex with polyanions negatively correlate with charge-related properties of the proteins such as isoelectric point and surface charge density and hydrophobicity of the polyanions.

Coulombic and noncoulombic effect of polyanions on cytochrome c structure.

The properties of the complexes of ferricytochrome c with two different polyanions--poly(vinylsulfate) and poly(4-styrene-sulfonate)--with a comparable charge density but with the different size of the uncharged part of their molecules have been studied by means of optical spectroscopy, differential scanning colorimetry, and gel chromatography. Ferriccytochrome c formed a complex with the former one through coulombic interactions and remained in a native-like state. The addition of the second polyanion to a solution of ferric cytochrome c at a low ionic strength, pH 7.0, resulted in profound conformational change in the hydrophobic core of protein (opening of the heme crevice with a perturbation of the methionine 80-heme iron bond and the hydrophobic core of the protein). These may be understood as an involvement of noncoulombic (hydrophobic, H-bonding) interactions of the uncharged part of the polyanion molecule. Conformational changes and the observed shift in acidic transition from low spin to high spin state of ferric cytochrome c detected in the presence of the polyanions may have biological implication in understanding the origin of conformational changes in proteins induced in the course of their interaction with membrane lipids and membrane proteins.

Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH

The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pK(a) values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55+/-0.08 in the 620 nm band region and 5.47+/-0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions.

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and differential scanning calorimetry (DSC) to study the properties of novel bioengineered DNA aptamer dimers sensitive to fibrinogen (F) and heparin (H) binding sites of thrombin and compared them with canonical single stranded aptamer sensitive to fibrinogen binding site of thrombin (Fibri). The homodimer (FF) and heterodimer (FH) aptamers were constructed based on hybridization of their supported parts. CD results showed that both FF and FH dimers form stable guanine quadruplexes in the presence of potassium ions like those in Fibri. The thermal stability of aptamer dimers was slightly lower compared to those of canonical aptamers, but sufficient for practical applications. Both FF and FH aptamer dimers exhibited a potassium-dependent inhibitory effect on thrombin-mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.

Spectrophotometric detection of the interaction between cytochrome c and heparin

Heparin inhibits transport of electrons from reduced cytochrome c to cytochrome c oxidase. The effect is due to the interaction of heparin with cytochrome c. It has been observed that binding of heparin to the reduced or oxidized cytochrome c changes the spectrum of cytochrome c at the Soret region. Affinity chromatography of heparin in cytochrome c immobilized to thiol-Sepharose shows that commercial heparin is eluted in the low-affinity and high-affinity fractions. Both participate in the interaction with cytochrome c. Polylysine induces decay of the cytochrome c-heparin complex.

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c.

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.

Interaction of cytochrome c with zinc oxide nanoparticles

The influence of pH on the interaction between horse heart ferricytochrome c (cyt c) and zinc oxide nanoparticles (ZnO NPs) has been studied by a small angle scattering as well as UV-vis and FTIR spectroscopy. The observations showed that the optimal pH for the association of protein with nanoparticles is in pH range 5.0-8.0. Almost no significant change in structure and thermodynamic stability of cytochrome c after the association with 60 nm ZnO NPs was performed by UV-vis and by a circular dichroism spectroscopy.