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Preparation of neprosin I - unique aspartic protease with potential in structural biology, proteomics and treatment of gluten intolerance.
Pelechová, Kamila ; Man, Petr (advisor) ; Šmídová, Aneta (referee)
Proteases are being found in all realms from viruses and microorganisms to plants and animals, including humans. They are necessary for living organisms. They degrade proteins to peptides and amino acids, allowing organisms to better absorb nutrients. Proteases also have a regulatory function - they control the cell cycle, they are responsible for converting precursors to biologically active substances, they are part of signaling cascades. Thanks to their properties, proteases are used for therapeutic purposes but found their used also in industry and research. This work deals with the production of recombinantly prepared neprosin - a newly discovered prolyl endopeptidase isolated from the carnivorous plant Nepenthes ventrata. It is an acidic protease with potential for use in structural biology, proteomics and medicine. Due to its ability to cleave primarily C-terminal proline residues, neprosin become a promising option for celiac enzyme therapy. (In Czech)
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Characterization and application of proline-selective proteases.
Portašiková, Jasmína Mária ; Man, Petr (advisor) ; Kádek, Alan (referee)
Peptide bond formed by proline is resistant to most known proteases, therefore the discovery and isolation of proteases cleaving after this amino acid opens up new possibilities not only for protein characterization but also for industrial, food or pharmaceutical aplications. Aspergillus niger prolyl endopeptidase (AnPEP) is a proline-selective protease that is commercially available in a variety of products. In this bachelor thesis cleavage preferences of proteases AnPEP (Clarity Ferm) and ProAlanase (Promega) were characterized and compared. The effect of different conditions on the specificity of proteases, while cleavaging the mixture of model proteins was examined within this work. AnPEP cleavage preferences were characterized on complex protein mixtures and its immobilized form was tested as well. [IN CZECH] Keywords: ProAlanase, AnPEP, H/D exchange, structural proteomics, imobilized proteases [IN CZECH]
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Characterization of protein structures using chemical cross-linking and mass spectrometry.
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee) ; Hernychová, Lenka (referee)
Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.
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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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Preparation of neprosin I - unique aspartic protease with potential in structural biology, proteomics and treatment of gluten intolerance.
Pelechová, Kamila ; Man, Petr (advisor) ; Šmídová, Aneta (referee)
Proteases are being found in all realms from viruses and microorganisms to plants and animals, including humans. They are necessary for living organisms. They degrade proteins to peptides and amino acids, allowing organisms to better absorb nutrients. Proteases also have a regulatory function - they control the cell cycle, they are responsible for converting precursors to biologically active substances, they are part of signaling cascades. Thanks to their properties, proteases are used for therapeutic purposes but found their used also in industry and research. This work deals with the production of recombinantly prepared neprosin - a newly discovered prolyl endopeptidase isolated from the carnivorous plant Nepenthes ventrata. It is an acidic protease with potential for use in structural biology, proteomics and medicine. Due to its ability to cleave primarily C-terminal proline residues, neprosin become a promising option for celiac enzyme therapy. (In Czech)
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Preparation of Oryzasin 1 for protein digestion in hydrogen/deuterium exchange.
Šintáková, Kristýna ; Man, Petr (advisor) ; Jurnečka, David (referee)
Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) is an increasingly popular technique in structural biology. Its spatial resolution strongly depends on the efficiency of the fragmentation or proteolytic cleavage of the studied protein. Therefore, it is desired to search for new proteases that would not only be able to digest the protein of interest under the HXMS conditions, but also to provide the best possible coverage of the protein sequence. Finding optimal conditions for production of Oryzasin 1 aspartate protease for its potential use in HXMS experiments was done in this thesis. Selected production clones were selected from available plasmids, the identity of the produced protein was verified by peptide mapping, and optimal production conditions were found. Based on these results, large-scale protein production and inclusion body isolation were undertaken. (In Czech) Keywords: Oryzasin 1, proteases, hydrogen/deuterium exchange coupled to mass spectrometry (HXMS)
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Analysis of Histone Deacetylase 6/Kinesin Interactions
Nedvědová, Jana ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Intracellular transport is provided by two major types of molecular motors kinesins and cytoplasmic dynein. Kinesin-1 is a molecular motor that transports molecules and organelles along microtubule tracks anterogradely. Specific protein-protein interactions are required to activate kinesin-1 as the free kinesin exist in an autoinhibited state. The activation of kinesin-1 induces its conformational change, enables microtubule binding and ATP hydrolysis necessary for the directional cargo transport. HDAC6 is a multifunctional protein composed of several domains. It plays an important role in many microtubule dependent processes as HDAC6 is a major tubulin deacetylase. It has been shown that HDAC6 manipulation (inhibition/genetic ablation) affects transport along microtubules but the exact mechanisms are unknown. The effect can be caused either by deacetylation microtubules or direct interaction with molecular motors. This thesis is focused on characterization of interactions between kinesin-1 and HDAC6 that have not been described so far. To this end, we expressed and purified various constructs of kinesin-1 and HDAC6 and tested their interactions by microscale thermophoresis (MST) and hydrogen deuterium exchange (HDX) to determine affinity and interaction sites, respectively. MST data revealed that...
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Characterization of protein structures using chemical cross-linking and mass spectrometry.
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee) ; Hernychová, Lenka (referee)
Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.
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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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