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Studies on interactions between natural killer cell lectin receptors and their protein ligands.
Hernychová, Lucie ; Novák, Petr (advisor) ; Drbal, Karel (referee)
NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.
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Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
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Investigation of the β-N-Acetylhexosaminidase Stucture from Penicillium oxalicum.
Krunclová, Tereza ; Novák, Petr (advisor) ; Hýsková, Veronika (referee)
in English β-N-Acetylhexosaminidase (EC 3.2.1.52) is exoglycosidase, which exhibits the unique properties in the filamentous fungi. Enzyme from these organisms are dimeric, inducible and secreted extracelluary. It is expresed as preproprotein, consists of a signal sequence, a large propeptid and a catalytic subunit. Although the enzyme is widely distributed, its structure differs in varies organisms. Bacteria have only monomeric hexosaminidase. Human enzymes are dimeric as well as fungal, but only hexosaminidase from filamentous fungi have the catalytic subunit noncovalently associated with the propeptide. Propeptide is a essential for the enzyme activity. It exists a homologues model of the catalytic subunit of β-N-acetylhexosaminidase from Penicillium oxalicum, but the structure of the propeptide has not yet been solved. The first part of this diploma thesis deals with the optimization of production and purification conditions. The second part deals with structural studies of β-N-acetylhexosaminidases from the filamentous fungi Penicillium oxalicum CCF 3438. These studies were carried out using chemical cross-linking and high resolution mass spectrometry. The combination of these methods revealed region of the noncovalent interaction of the catalytic subunit with the propeptide.
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Investigation of the β-N-Acetylhexosaminidase Stucture from Aspergillus oryzae
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Ryšlavá, Helena (referee)
in English β-N-acetylhexosaminidase (EC 3.2.1.52) belongs to exoglycosidase, and is one of the most abundant enzymes found in organisms from bacteria to human. The fungal β-N-acetylhexosaminidase from Aspergillus oryzae is composed of propeptide and catalytic domain. The propeptide is noncovalently associated with the catalytic domain of the enzyme. Propeptide is essential for the enzyme activity. While the structure of the catalytic domain was desidned by homology modeling, the structure of the propeptide has not been resolved yet. In this study, the position where the propeptide is associated with the catalytic domain, was uncovered. Presented work consists of two parts. First part deals with optimization of production conditions, purification and crystallization of β-N-acetylhexosaminidase from the filamentous fungi Aspergillus oryzae. Second part is devoted to the study of interaction between propeptide and catalytic domain, which was characterized by chemical cross-linking and high-resolution mass spectrometry. It was found that the structural changes of the catalytic domain depend on the presence of the propeptide molecule. Moreover the region of propeptide-catalytic domain interaction was revealed.
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