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Comparison of apo- and holoforms of the transcription factor "Bach1"
Vávra, Jakub
Hemoproteins represent very important components of many living organisms. Participation in the processes of oxygen transport and storage, electron transport or enzymatic catalysis of reactions involving oxygen or hydrogen peroxide are commonly known functions of hemoproteins. Recently, there has been discovered a new group of hemoproteins. The main feature of this new group of proteins is their ability to detect changes in heme concentration (heme-responsive proteins) or changes in diatomic gas concentration (gas-responsive heme-containing sensor proteins) in their vicinity. Detection of these concentration changes generates signals that induce structural changes of the respective sensor proteins. Finally, the structural changes of the respective sensor proteins affect their functions or activities. The subject of this diploma thesis is the preparation and characterization of the eukaryotic heme sensor Bach1. We especially focused on the ability of Bach1 to bind heme molecules and on the comparison of various Bach1 properties in its apoform and holoform. Determination of the exact amount of heme molecules that specifically interact with heme sensor Bach1 represents very important part of this thesis. We also studied the effect of different redox states of heme iron and the presence of interaction...
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Comparison of apo- and holoforms of the transcription factor "Bach1"
Vávra, Jakub ; Martínková, Markéta (advisor) ; Brynychová, Veronika (referee)
Hemoproteins represent very important components of many living organisms. Participation in the processes of oxygen transport and storage, electron transport or enzymatic catalysis of reactions involving oxygen or hydrogen peroxide are commonly known functions of hemoproteins. Recently, there has been discovered a new group of hemoproteins. The main feature of this new group of proteins is their ability to detect changes in heme concentration (heme-responsive proteins) or changes in diatomic gas concentration (gas-responsive heme-containing sensor proteins) in their vicinity. Detection of these concentration changes generates signals that induce structural changes of the respective sensor proteins. Finally, the structural changes of the respective sensor proteins affect their functions or activities. The subject of this diploma thesis is the preparation and characterization of the eukaryotic heme sensor Bach1. We especially focused on the ability of Bach1 to bind heme molecules and on the comparison of various Bach1 properties in its apoform and holoform. Determination of the exact amount of heme molecules that specifically interact with heme sensor Bach1 represents very important part of this thesis. We also studied the effect of different redox states of heme iron and the presence of interaction...
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Characterization of selected properties of model heme-containing sensor proteins
Fojtík, Lukáš ; Martínková, Markéta (advisor) ; Svášková, Dagmar (referee)
Heme sensor proteins are the fourth group of hemoproteins. In this group of hemoproteins heme plays an important role in signalization. Dissociation and/or association of heme detecting proteins serves as an important physiological function in regulation of enzyme activity or gene expression. In this bachelor thesis all the actual knowledge about selected forms of eukaryotic heme sensor proteins previously published in scientific articles are summarized. The experimental part of this bachelor thesis is focused on preparation of recombinant protein heme regulated inhibitor (HRI) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). Firstly the preparation of the plasmids with genes HRI and eIF2α was conducted. In the next step these proteins were prepared in prokaryotic system formed by E. coli BL-21(DE3). The final sample of HRI (7,7 μM in total volume 400 µl and 60 % of homogenity) and the final sample of eIF2α (51,3 μM in total volume 400 µl and 80 % of homogenity) were obtained by the purification process. The study of thermal stability of these samples provided important informations on appropiate storage and manipulation with them in further experiments. Key words: heme-base sensors, heme, kinase, tranduction of signal, isolation of plasmids, prokaryotic expresion,...
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