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Preparation of the 14-3-3 Protein Binding Partners for Structural Studies.
Kopecká, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Tyrosine hydroxylase belongs to the group of hydroxylases of aromatic acids and catalyzes a key step in the biosynthesis of catecholamine neurotransmitters. The tyrosine hydroxylase possesses the homotetrameric structure and contains three structural domains: the N-terminal regulatory domain, the catalytic domain and the C-terminal tetramerization domain. The activity of tyrosine hydroxylase is regulated by phosphorylation and through the regulation of its expression. Phosphorylation at Ser-19 induces binding of the 14-3-3 protein, which affects the structure of the regulatory domain and protects it against both dephosphorylation and degradation. Since the structure of the regulatory domain is still unknown, we decided to perform its structural characterization using NMR techniques. First, the expression and purification protocol of the regulatory domain of tyrosine hydroxylase was optimized. The protein was expressed as a His-tag fusion protein and its purification is composed from two steps: the chelating chromatography and the size-exclusion chromatography. The dynamic light scattering and the 1 H nuclear magnetic resonance were used to verify its monodispersity, and hence its suitability for further experiments.
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Molecular mechanism of the neutral trehalase Nth1 regulation
Kopecká, Miroslava ; Obšilová, Veronika (advisor) ; Bařinka, Cyril (referee) ; Pompach, Petr (referee)
The yeast enzyme neutral trehalase (Nth1, EC 3.2.1.28) from the Saccharomyces cerevisiae helps these organisms to survive adverse living conditions. Nth1 hydrolyses a storage and protective disaccharide trehalose into two molecules of glucose. The activity of this enzyme is regulated by PKA phosphorylation, Ca2+ binding and the yeast 14-3-3 protein (Bmh1) binding. Ca2+ binds to the Ca-binding domain located within N-terminus of Nth1 and contains so called EF-hand motif (D114 TDKNYQITIED125 ) which is highly conserved among many Ca-binding proteins. The main aim of this project was to reveal the structural basis of the Bmh1- and calcium-dependent activation of Nth1. Other goals were to solve the structure of Nth1 itself and the structure of its complex with Bmh1. To reveal how the calcium regulates the Nth1 activity we prepared twelve mutant forms of Nth1 using site directed mutagenesis. These mutations were located within the region of EF-hand motif and its close vicinity. We estimated the enzymatic activity of all these mutants in the presence of Bmh1 and/or Ca2+ . The ability of Nth1 to form stable complexes with Bmh1 was verified using the native polyacrylamide gel electrophoresis and analytical ultracentrifugation. The impact of mutations on the structure and properties of Nth1 was tested using...
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Service quality monitoring in libraries (focused on university libraries)
Kopecká, Miroslava ; Drobíková, Barbora (advisor) ; Stöcklová, Anna (referee)
This master thesis deals with service quality monitoring in libraries and it focuses especially on the situation in university libraries. There are foreign tools described in this thesis, namely the American LibQUAL+® which uses user satisfaction surveys, and German Bibliothekxindex which compares libraries according to recieved statistical data. From the Czech environment there is an initiative of the National Library of the Czech Republic described, "Benchmarking of Libraries", which is based on the German Bibliotheksindex but it is not applicable in university libraries. The thesis includes a self made suggestion on how to monitor service quality according to both of the foreign tools which was also applied to a selected university library. The results of this research indicate that these foreign tools are applicable in the Czech Republic and that the selected university library provides services which its users are overall very satisfied with.
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Preparation of the 14-3-3 Protein Binding Partners for Structural Studies.
Kopecká, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Tyrosine hydroxylase belongs to the group of hydroxylases of aromatic acids and catalyzes a key step in the biosynthesis of catecholamine neurotransmitters. The tyrosine hydroxylase possesses the homotetrameric structure and contains three structural domains: the N-terminal regulatory domain, the catalytic domain and the C-terminal tetramerization domain. The activity of tyrosine hydroxylase is regulated by phosphorylation and through the regulation of its expression. Phosphorylation at Ser-19 induces binding of the 14-3-3 protein, which affects the structure of the regulatory domain and protects it against both dephosphorylation and degradation. Since the structure of the regulatory domain is still unknown, we decided to perform its structural characterization using NMR techniques. First, the expression and purification protocol of the regulatory domain of tyrosine hydroxylase was optimized. The protein was expressed as a His-tag fusion protein and its purification is composed from two steps: the chelating chromatography and the size-exclusion chromatography. The dynamic light scattering and the 1 H nuclear magnetic resonance were used to verify its monodispersity, and hence its suitability for further experiments.
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