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Study of intermolecular interactions by isothermal titration calorimetry
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
Calorimetric methods are used to studying the mechanism of regulation and control of biological processes at the molecular level. Isothermal titration calorimetry (ITC) is used for monitoring the heat exchange of molecular interactions in an environment with constant temperature. This method is also preferred because of its ability of direct measurement of thermodynamic parameters associated with complex formation. ITC measures mostly the energetics of biochemical reactions or molecular interactions protein-protein, protein-ligand, or enzyme-substrate. Main goals of this bachelor thesis were: (I) to acquire working skills with nano isothermal titration calorimeter; (II) to prepare the DNA-binding domain of human transcription factor FOXO4 (FOXO4-DBD); and (III) to study thermodynamic aspects of the interaction between FOXO4-DBD and the double-stranded DNA containing specific binding motif. FOXO4 protein is one of the four members of a "O" subgroup of forkhead transcription factors FOX, which play an important role in many cellular processes including stress resistance, age control and oncogenesis. Forkhead DNA- binding domain recognizes sequences containing the core motif 5'-(A/C)AA(C/T)A-3' with the binding stoichiometry 1:1. Results of this bachelor thesis show that FOXO4-DBD binds the...
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Molecular mechanism of the neutral trehalase Nth1 regulation
Alblová, Miroslava
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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Molecular mechanism of the neutral trehalase Nth1 regulation
Alblová, Miroslava
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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Study of the posttrans lation al modifications of phosducin
Šimůnek, Jiří ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
The aim of this diploma thesis was to study the role of posttranslational modifications of phosducin and their role in the interaction with the 14-3-3 protein as well as the influence of the complex formation on these modifications. Phosducin is a 33kDa protein commonly present in photoreceptor cells of the retina as well as other tissues. Despite many experiments, its physiological functions are still elusive. It has been speculated that fosducin plays an important regulatory role in visual phototransduction pathway, regulation of blood pressure and expression of G-proteins. The phosducin function is regulated through binding to the 14-3-3 protein, a regulatory protein involved in many biochemical processes. Phosducins binding to 14-3-3 protein requires phosphorylation of two serine residues Ser-54 and Ser-73 within the N-terminal domain of phosducin. However, the role of the 14-3-3 protein binding in the regulation of phosducin function is still unclear. In this diploma thesis proteins 14-3-3ζ∆C and phosducin (mutation Q52K) were successfully expressed and purified. The effect of the complex formation on phosducin posttranslational modifications was investigated using limited proteolysis and dephosphorylation. These experiments revealed that the complex formation significantly slowed down both...
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Study of interaction between phosducin and the 14-3-3 protein
Šimůnek, Jiří ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
The main goal of this bachelor thesis is to study the interaction between phosducin and the 14-3-3 protein which are involved in the regulation of visual signal transduction in vertebrate retina. This process is mediated by G-protein signaling pathway and is regulated by several proteins including phosducin depending on light intensity. Phosducin, a 33kDa protein, is expressed in many tissues mainly in photoreceptor cells of the retina. The visual signal transduction and its amplification are regulated through inhibition of the G-protein (transducin) function. The signal transduction involves a dissociation of heterotrimeric transducin (Gtαβγ) to the α subunit (Gtα) and the βγ complex (Gtβγ). The signalling is terminated by their reassociation back to the Gtαβγ complex. The phosducin binds to Gtβγ and thus prevents the formation of the Gtαβγ heterotrimer. This decreases the amount of functional Gtαβγ complexes and suppresses the signal transduction. If the signal transduction needs to be amplified (e.g. during the night) then phosducin is phosphorylated and this inhibits its interaction with Gtβγ. The phosphorylated phosducin is bound to the 14-3-3 protein. However, the role of the 14-3-3 protein in the regulation of phosducin is still unclear. The 14-3-3 protein is a 28kDa protein which is...
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Study of intermolecular interactions by isothermal titration calorimetry
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
Calorimetric methods are used to studying the mechanism of regulation and control of biological processes at the molecular level. Isothermal titration calorimetry (ITC) is used for monitoring the heat exchange of molecular interactions in an environment with constant temperature. This method is also preferred because of its ability of direct measurement of thermodynamic parameters associated with complex formation. ITC measures mostly the energetics of biochemical reactions or molecular interactions protein-protein, protein-ligand, or enzyme-substrate. Main goals of this bachelor thesis were: (I) to acquire working skills with nano isothermal titration calorimeter; (II) to prepare the DNA-binding domain of human transcription factor FOXO4 (FOXO4-DBD); and (III) to study thermodynamic aspects of the interaction between FOXO4-DBD and the double-stranded DNA containing specific binding motif. FOXO4 protein is one of the four members of a "O" subgroup of forkhead transcription factors FOX, which play an important role in many cellular processes including stress resistance, age control and oncogenesis. Forkhead DNA- binding domain recognizes sequences containing the core motif 5'-(A/C)AA(C/T)A-3' with the binding stoichiometry 1:1. Results of this bachelor thesis show that FOXO4-DBD binds the...
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