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Singlet oxygen quenching is sensitive to the exposure of heme to the solvent.
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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... more
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.
Research Interests: Engineering, Chemical Engineering, Chemistry, Polymers, Comparative Study, and 15 moreMedicine, Biological Sciences, Heparin, Differential scanning calorimetry, Lysozyme, Cytochrome C, Material, Partition Coefficient, CHEMICAL SCIENCES, Aqueous Solution, Database, Polyelectrolyte, Biomacromolecules, Polystyrenes, and Isoelectric point
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Fluorescence and structure of acridin-9-ylthioureas with primary amino rest and their Smethylated products have been studied. The synthesized 1-(acridin-9-yl)-3-alkyl-S-methylisothiuronium iodides exhibit about one order increased... more
Fluorescence and structure of acridin-9-ylthioureas with primary amino rest and their Smethylated products have been studied. The synthesized 1-(acridin-9-yl)-3-alkyl-S-methylisothiuronium iodides exhibit about one order increased fluorescence in comparison with corresponding thioureas. The structure of products obtained by methylation reactions was confirmed by NMR techniques including PDQF-COSY, selective INEPT, NOE difference experiments, and quantumchemical calculations (AM1, ab initio). A free rotation of 9-substituents in relation to acridine skeleton has been found. The conformers of acridinylisothioureas are in E-configuration relative to the C==N bond with more favourable imino (C-9==N) tautomeric structure.
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Research Interests: Chemistry, Free Radicals, Photochemistry, Mitochondria, Medicine, and 15 moreFree Radical, Peroxidase, Animals, Cytochrome C, Cytochrome c oxidase, Catalase, Cattle, Hydrogen Peroxide, Myocardium, Oxidative Damage, Electron Paramagnetic Resonance Spectroscopy, Biochemistry and cell biology, Cardiolipin, Free Radical Biology, and Rate Constant
NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature,... more
NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e. at approximately 70 degrees C for T. thermophilus. At low temperatures, the kcat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents. The catalytic rate of NADH oxidase increases in the presence of urea. At concentrations of 1.0-1.3 m urea it reaches 250% of the activity in the absence of urea, at 20 degrees C. At higher urea concentrations the enzyme activity is inhibited. The urea-dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme. Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme. This is not related to the global structure of NADH oxidase. The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin-dependent oxidases. A comparison of the crystal structure with the activation thermodynamic parameters, deltaH* and TdeltaS*, obtained from the temperature dependence of kcat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring. The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.
Research Interests: Thermodynamics, Chemistry, Catalysis, Kinetics, Medicine, and 15 moreEnzyme Kinetics, Circular Dichroism, Temperature, Urea, Tryptophan, Active site, Time Factors, Multienzyme complexes, Protein Conformation, Flavins, Biochemistry and cell biology, Dimerization, Fluorescence quenching, binding sites, and Medical biochemistry and metabolomics
ABSTRACT
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<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.©... more
<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.© 2007 The Author(s) Tripeptide motifs are highlighted [GGQ and SP(F/Y); grey ovals] as well as the possible hinge regions for movement of domain III (black circles). () Overlay of the ribbon structures of (red) and (green) RF2 structure models. () Model of an elongated, ribosomal bound RF2 as determined by cryo-electron microscopy (,). () Alignment of primary structures and hydropathy plot of and RF2s.
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<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.©... more
<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.© 2007 The Author(s) Upper panel: protein ellipticity in the far-UV region of CD at 220 nm. Scanning rate was 90 K/h, proteins concentration 1.5 μM in 20 mM sodium cacodylate, pH 7.2, 300 mM NaCl, and 1 mM β-mercapthoethanol. Ellipticity [Θ] is given in 10 deg cm/dmol units. Lower panel: calorimetric scans of RFs (thick lines, and ) obtained upon subtraction of baselines. Second heating scans are shown as dashed () and dotted lines (). The scan rate was 90 K/min, the protein concentrations 24 μM. Curve fits of thermal denaturation based on the single and multiple two state processes are shown as solid lines. relative composition of ΔASA of interfaces between domain I and domains II·III·IV of RF2s from and , respectively—polar amino acids (white box), nonpolar amino acids (grey box).
Research Interests: Chemistry and Calorimetry
Research Interests: Biochemistry, Physiology, Chemistry, Kinetics, Medicine, and 15 moreCircular Dichroism, Medical Physiology, Enzyme, Multienzyme complexes, Enzyme activity, Protein Conformation, Energy Transfer, Protein Binding, Adenine, Flavins, Conformational Change, Histidine, Flavoprotein, Dimerization, and binding sites
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Research Interests: Chemistry, Crystallography, Protein Folding, Polymers, Medicine, and 12 moreBiological Sciences, Circular Dichroism, Differential scanning calorimetry, Biopolymers, Animals, Cytochrome C, Horses, Spectrophotometry, Molten Globule, CHEMICAL SCIENCES, Protein Secondary Structure, and Hydrogen-Ion Concentration
... Kamil Tóth a , Erik Sedlák a , b , Andrej Musatov b and Gabriel oldák c , Corresponding Author Contact Information , E-mail The Corresponding Author. a Department of Biochemistry, Faculty of Sciences, PJ afárik University, Koice,... more
... Kamil Tóth a , Erik Sedlák a , b , Andrej Musatov b and Gabriel oldák c , Corresponding Author Contact Information , E-mail The Corresponding Author. a Department of Biochemistry, Faculty of Sciences, PJ afárik University, Koice, Slovakia. ...
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Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but... more
Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due...
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Fluorescence and structure of acridin-9-ylthioureas with primary amino rest and their Smethylated products have been studied. The synthesized 1-(acridin-9-yl)-3-alkyl-S-methylisothiuronium iodides exhibit about one order increased... more
Fluorescence and structure of acridin-9-ylthioureas with primary amino rest and their Smethylated products have been studied. The synthesized 1-(acridin-9-yl)-3-alkyl-S-methylisothiuronium iodides exhibit about one order increased fluorescence in comparison with corresponding thioureas. The structure of products obtained by methylation reactions was confirmed by NMR techniques including PDQF-COSY, selective INEPT, NOE difference experiments, and quantumchemical calculations (AM1, ab initio). A free rotation of 9-substituents in relation to acridine skeleton has been found. The conformers of acridinylisothioureas are in E-configuration relative to the C==N bond with more favourable imino (C-9==N) tautomeric structure.
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<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.©... more
<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.© 2007 The Author(s) RF2 from (open circles) and (solid circles) were measured at 1.5 μM protein concentration in 20 mM sodium cacodylate, pH 7.2 containing 300 mM NaCl and 1 mM β-mercaptoethanol at 20°C. Curves were generated by fitting the experimental data to .
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Cytochrome c (cyt c), in addition to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a critical role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads... more
Cytochrome c (cyt c), in addition to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a critical role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads to a suicide inactivation of the protein, which is accompanied by heme destruction and covalent modification of numerous amino acid residues. Although steady-state reactions of cyt c with an excess of hydrogen peroxide represent non-physiological conditions, they might be used for analysis of the first-modified amino acid in in vivo. Here, we observed oxidation of tyrosine residues 67 and 74 and heme as the first modifications found upon incubation with hydrogen peroxide. The positions of the oxidized tyrosines suggest a possible migration pathway of hydrogen peroxide-induced radicals from the site of heme localization to the protein surface. Analysis of a size of folded fraction of cyt c upon limited incubation with hydrogen peroxide indicates that the early oxidation of amino acids triggers an accelerated destruction of cyt c. Position of channels from molecular dynamics simulation structures of cyt c points to a location of amino acid residues exposed to reactive oxidants that are thus more prone to covalent modification.
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Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme... more
Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0-4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-in...
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Interaction of polyanion poly(vinylsulfate) with oxidized cytochrome c (cyt c) significantly affects the protein main characteristics. One of them, pKa value of acidic transition, was shifted from an apparent pKa value 2.5 (typical for... more
Interaction of polyanion poly(vinylsulfate) with oxidized cytochrome c (cyt c) significantly affects the protein main characteristics. One of them, pKa value of acidic transition, was shifted from an apparent pKa value 2.5 (typical for cyt c in low ionic strength solvent) to approximately 5.20 +/- 0.15 upon polyanion binding to the protein, pointing to a likely involvement of histidines 26 and/or 33 in the protein acidic transition in complex with the polyanion. The acidic transition followed at 6 different wavelengths all over circular dichroism spectrum, monitoring different parts of the protein structure, revealed basically two-state character process. Only ellipticity at 262 nm indicated a low-cooperative pH-induced conformational transition in heme region with an apparent pKa approximately 4.34 +/- 0.25 in accordance with absorbance change at 620 nm. Polyanion also interacts with chemically-denatured (in the presence of 9 mol/l urea) state of the protein as it follows from stab...
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Von Willebrand Factor (VWF) initiates platelet adhesion at sites of vessel injury via the binding of platelet glycoprotein (GP) Ib-IX-V surface receptor to the VWF A1 domain. Congenital mutations occurring in the A1 domain result in the... more
Von Willebrand Factor (VWF) initiates platelet adhesion at sites of vessel injury via the binding of platelet glycoprotein (GP) Ib-IX-V surface receptor to the VWF A1 domain. Congenital mutations occurring in the A1 domain result in the clinical bleeding diathesis of several subtypes of Type 2 Von Willebrand Disease. These mutations can either enhance VWF-platelet interactions (Type 2B) or cause a defect in this interaction (Type 2M). We hypothesize that these functional types of mutations could differentially alter VWF-platelet interactions in part by affecting the intrinsic stability of the A1 domain structure and consequently, a conformational transition induced by these interactions. In this study, we have characterized the thermodynamics of unfolding the A1 domain containing the Type 2B mutations (R1306Q, R1308L, and I1309V), the Type 2M mutation (G1324S) and a blocked disulfide variant of A1, (reduced and carboxyamidated, RCAM A1), that is representative of the Type 2M disulfi...
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The structure, fluorescence, biological properties and S(N)-methylation reactions of ten 1,1-alkyl/aryl-disubstituted 3-(acridin-9-yl)thioureas 4 have been studied. Various reaction conditions allowed to obtain corresponding S-methyl 5... more
The structure, fluorescence, biological properties and S(N)-methylation reactions of ten 1,1-alkyl/aryl-disubstituted 3-(acridin-9-yl)thioureas 4 have been studied. Various reaction conditions allowed to obtain corresponding S-methyl 5 and S,N-dimethyl derivatives 6 in good yields. Structure and stereochemistry of the synthesized products are demonstrated by ab initio quantum chemical calculations and NMR techniques including PDQF-COSY, selective INEPT and NOE-difference experiments. Remarkable upfield 13C shifts of resonance signals of carbons C-4a, C-10a adjacent to acridine N-10 are characteristic of hydroiodides in contrast to free bases. Z configuration in isothioureas 7 with secondary amino rest in relation to E configuration of isothioureas with primary amino rest is discussed. Of the obtained products, some isothiourea salts 6 exhibit more than 2 orders of magnitude higher intensity of fluorescence, using 9-isothiocyanatoacridine as a standard. The obtained isothiourea hydro...
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<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.©... more
<b>Copyright information:</b>Taken from "Release Factors 2 from and : structural, spectroscopic and microcalorimetric studies"Nucleic Acids Research 2007;35(4):1343-1353.Published online 01 Feb 2007PMCID:PMC1849895.© 2007 The Author(s) CD spectra were measured in 2 mM sodium phosphate, pH 7.2, in a 1 mm path length cuvette. Concentrations of RF2 from (empty circles) and (solid circles) were 2.95 and 1.4 μM, respectively. The fitted curves are shown by solid lines.
Abstract Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c ) have been studied by the UV-VIS absorbance spectroscopy, intrinsic... more
Abstract Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c ) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The p K a value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased p K a (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c . The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.