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Recombinant expression of immunoglobulin superfamily receptor NKp30
Tůmová, Lucie ; Kavan, Daniel (advisor) ; Šulc, Miroslav (referee)
1 NK cells belong to lymphocytes, which are able to kill some tumor cells and virus-infected cells, produce cytokines and participate in the formation of the adaptive immune response. This bachelor thesis deals with human receptor of NK cells, specifically NKp30. This protein belongs to the group of activating receptors which can activate the cytotoxic function of NK cells. Its three-dimensional structure and the interaction between NKp30 and its physiological ligand B7-H6 has been recently resolved. Howewer the interaction with the other ligands, BAT3 and pp65, remains unknown. The aim of the work was optimalization of expression of the NKp30 receptor extracellular domain in prokaryotic expression system. The structural studies with ligands of this protein may provide further details on the interaction between the receptor NKp30 and ligands BAT3 and pp65. (In Czech)
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Protein-protein interaction mapping of cytochrome P-450 by methods using /chemical modification and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor)
Mapping of protein-protein interactions in the system of cytochrom P-450 by chemical modification and mass spectrometry Abstract Cytochromes P-450 (P450s) belong to haemoprotein superfamily and they are responsible for metabolism of a wide variety of compounds, among others many drugs and carcinogens. P450s serve as the terminal oxidases in the mixed function oxidase system in cooperation with a redox partner NADPH: cytochrome P450 reductase (CPR) providing input of two electrons to the reaction cycle of P450. The CPR can be substituted by other redox partner of P450, cytochrome b5 (cyt b5), to deliver the second electron. Three dimensional structure of P450 is required in order to fully understand its reaction mechanism. At the present time, a homology model of cytochrome P-450 2B4 (CYP 2B4) is available in our laboratory. In this study, the mapping of interaction domain between CYP 2B4 and cyt b5 employing a crosslinking agent EDC to form amide bonds between close complementary charged amino acid side chains was the first goal. We have identified five interacting amino acid pairs in total using mass spectrometry (MS). The second research interest was to verify and refine the CYP 2B4 model using a photoaffinity labelling with N-(p-azidobenzyl)-N-methyl-p-aminophenylamine probe. This photoreactive probe is...
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Purification and characterization of selected enzymes from Rhizobium radiobacter R89 catalysing oxidation/reduction reactions of C-hydroxylated morphine skelets.
Zahradník, Jiří ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
The Rhizobiaceae bacteria are known for its varied nitrogen metabolism: especially its nitrogen fixation, so called diazotrofia, its capability of symbiosis with plants, or on the contrary plant pathogenesis (genus Agrobacterium). Rhizobium radiobacter R89 (stored in Czech Collection of Microorganism, in Brno as R89-1) is bacteria strain capable of codeine and morphine biotransformation to whole spectrum of pharmacologically significant derivates. This work is focused on purification and characterization of two selected enzymes that catalyze oxidation-reduction reactions, the crucial beginning of morphine skelets biodegradation. At the first, the biotransformational potential of the strain was evaluated and biochemical, molecular biological and bioinformatical approaches were employed to purify the responsible enzymes. Considering unsuccessful purification from Rhizobium radiobacter R89-1, the proteins structural genes were cloned and heterologously expressed in bacterial system and afterward characterized. Found protein sequence and enzyme characterization (basic kinetic measurements, substrate specificity and thermo- stability) have revealed different origin of the enzymes and clarify reasons why the original purification procedure was not successful.
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Conformational changes of cytochrome c in acid nedia
Plutinský, Martin ; Hudeček, Jiří (advisor) ; Šulc, Miroslav (referee)
One part of my work was focused on literature review within the conformational changes of cytochrome c under various circumstances. Main goal of these studies is to clarify the importance of conformational changes of cytochrome c as the cell death messenger. Cytochrome c binds to cardiolipin- rich inner mitochondrial membrane and a molten globule-like intermediate is supposed to play still unknown role in cell death start. Cytochrom c is also common model protein in a "simple" protein folding problem. It is simple, small molecule and easy to obtain protein that can be studied by a broad variety of techniques. The second part of my work presents conformational study of horse heart ferricytochrome c under acidic conditions with addition of inorganic salts. These conformational changes were monitored using UV/VIS spectrophotometry in four areas of absorption spectrum - aromatic amino acids absorption area and so called Soret band, Q-band and CT band. Derivative of these spectra were used in order to obtain higher-resolution informations.
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Kinetic properties of β-N-acetylhexosaminidase from tobacco plants
Valenta, Robert ; Ryšlavá, Helena (advisor) ; Šulc, Miroslav (referee)
β-N-acetylhexosaminidase from tobacco leaves (Nicotiana tabacum L.) was partially purified to final specific activity 1,72 µmol . min-1 . mg-1 using p-nitrofenyl-β-N- acetyl-D-glucosaminide as substrate. The enzyme exhibited one band after both isoelectric focusing and native electrophoresis. Molecular mass of native enzyme was determined by gel chromatography (MR 275000) and native electrophoresis (MR 285000). Isoelectric point pI 5.4 was determined by isoelectric focusing. Activity of β-N-acetylhexosaminidase was measured using substrates p-nitrofenyl-β-N-acetyl-D-galactosaminide, p-nitrofenyl-β- N-acetyl-D-glucosaminide, N,N'-diacetylchitobiose, p-nitrofenyl-N,N'- diacetylchitobioside and N,N',N''-triacetylchitotriose. For substrates N,N'- diacetylchitobiose, p-nitrofenyl-N,N'-diacetylchitobioside and N,N',N''-triacetylchitotriose an enzyme assay of β-N-acetylhexosaminidase using capillary zone electrophoresis was developed. Optimal pH and temperature of β-N-acetylhexosaminidase were determined with individual substrates, as well as products of hydrolysis. Activity of β-N- acetylhexosaminidase was highest using p-nitrofenyl-β-N-acetyl-D-glucosaminide as substrate and lowest using N,N',N''-triacetylchitotriose (35% in relative comparison). Maximum velocity and Michaelis constant of...
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Study on conformational changes in proteins using mass spectrometry.
Rosůlek, Michal ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
Some proteins and enzymes require presence of their specific ligand, cofaktor or prosthetic group for their activity. Binding of this specific molecule causes conformational changes, which permits to perform their function. In some occasions the identification of conformational changes is difficult. Using chemical cross-linking coupled with mass spectrometry perform complex tool for searching and low resolution visualization of this changes. The aim of this thesis is study of conformational changes induced by binding of calcium ion to calmodulin protein molecule. Calmodulin is a secondary intermediate messenger, which can interact with various proteins. This feature associates with wide dynamical range of calmodulin. Thus calmodulin is the suitable target for identifying conformational changes. After reaction of protein with chemical cross-linkers with different arm length (DSG and DSS) were products of reaction digested by trypsine. Formed linked peptides were separated by high-performance liquid chromatography and analysed followed mass spectrometry. Seven unique intramolecular cross-links were identified. Using isotope unlabeled cross-link reagents in the presence of Ca2+ in combination with using isotope labeled reagents in calcium free conditions we quantified formed lysine-lysine cross-links....
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Excitation of molecules by cold electrons
Šulc, Miroslav ; Horáček, Jiří (advisor)
Title: Excitation of molecules by cold electrons Author: Miroslav Šulc Department / Institute: Institute of Theoretical Physics, Charles University in Prague Supervisor of the doctoral thesis: prof. RNDr. Jiří Horáček, DrSc., Institute of Theoretical Physics, Charles University Abstract: Several methods for low energy collisional processes are investigated. In the first part, attention is especially devoted to examination of applicability of the R-matrix method combined with the Schwinger-Lanczos (SL) variational principle for potential scattering with long-range forces. Next sections deal with the development of the interaction correlation-polarization (CP) potential in the framework of the Dis- crete Momentum Representation (DMR) method on the grounds of the Local Density Approximation in the Density Functional Theory (DFT) context. Obtained results are then utilized in body-frame (BF), static exchange + polarization (SEP), calcula- tions within an analysis of experimental data for e−-N2 scattering comprising a part of a larger project addressing theoretical examination of rotational excitations of small molecules in the gas phase induced by electron impact. For N2, a new phenomenon consisting in suppression of backward cross-section below 95 meV is observed and con- sequently attributed to...
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Excitation of molecules by cold electrons
Šulc, Miroslav ; Horáček, Jiří (advisor) ; Čárský, Petr (referee) ; Čížek, Martin (referee)
Title: Excitation of molecules by cold electrons Author: Miroslav Šulc Department / Institute: Institute of Theoretical Physics, Charles University in Prague Supervisor of the doctoral thesis: prof. RNDr. Jiří Horáček, DrSc., Institute of Theoretical Physics, Charles University Abstract: Several methods for low energy collisional processes are investigated. In the first part, attention is especially devoted to examination of applicability of the R-matrix method combined with the Schwinger-Lanczos (SL) variational principle for potential scattering with long-range forces. Next sections deal with the development of the interaction correlation-polarization (CP) potential in the framework of the Dis- crete Momentum Representation (DMR) method on the grounds of the Local Density Approximation in the Density Functional Theory (DFT) context. Obtained results are then utilized in body-frame (BF), static exchange + polarization (SEP), calcula- tions within an analysis of experimental data for e−-N2 scattering comprising a part of a larger project addressing theoretical examination of rotational excitations of small molecules in the gas phase induced by electron impact. For N2, a new phenomenon consisting in suppression of backward cross-section below 95 meV is observed and con- sequently attributed to...
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Mass spectrometry in proteomics: structural biology and clinical applications
Pavlásková, Kateřina ; Šulc, Miroslav (advisor) ; Obšilová, Veronika (referee) ; Hernychová, Lenka (referee)
Mass spectrometry (MS) is a rapid, specific and very sensitive analytical method with a broad spectrum of proteomic applications such as protein identification and sequencing, 3D protein structure characterization or study of protein-protein interaction. The introduction of two ionization techniques in late 1980's that are able to ionize the large biomolecules such as proteins, oligosaccharides or nucleic acids with no or low fragmentation has started the rapidly expanding field of MS-based proteomics. The presented thesis was aimed at the application of mass spectrometric approaches to answer several proteomic questions. Firstly we have employed the chemical cross-linking in combination with MS analysis to solve the 3D structure and protein-protein interactions of three model systems: (1) homodimeric human regulatory protein 14-3-3, (2) model of 14-3-3 and regulatory domain of tyrosine hydroxylase, and (3) system of two membrane proteins, cytochrome P450 2B4 and cytochrome b5, involved in xenobiotics biotransformation. This approach works in aqueous solutions under physiological conditions and thus preserves native structure of the investigated proteins. The second part of the thesis was focused on MS identification of proteins/peptides in fungal spores of Aspergillus and Pseudallescheria...
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