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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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Preparation and characterization of a bacterial protein YddV, a globin-coupled oxygen sensor diguanylate cyclase
Křížová, Věra ; Martínková, Markéta (advisor) ; Man, Petr (referee)
Heme-containing proteins are very important for proper function of various organisms, both prokaryotic and eukaryotic. The most well-known ones are myoglobin, hemoglobin and various peroxidases and cytochromes. The group of heme-containing proteins was recently extended by a new type of proteins, which are called heme-containing sensor proteins. These proteins consist of two domains, sensor domain, which is able to detect a signal (either a molecule of heme or a molecule of gas), and functional domain, which is often an enzyme or a transcriptional factor. Activity of the functional domain depends on the concentration of signal molecule, which binds to the sensor domain, which then causes change of its structure. Consequently, change of structure of the sensor domain also leads to change of structure of the functional domain. Heme-containing sensor proteins can be further divided into two smaller groups: heme sensor proteins and gas sensor proteins. Heme sensor proteins share a lot of features, for example binding of heme to a thiolate residue from cysteine, CP motif, etc. Gas sensor proteins detect a molecule of gas, especially oxygen, carbon monoxide and nitrogen monoxide. There have already been conducted various studies focusing on heme-containing sensor proteins, however, the mechanism of signal...
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Important roles of heme as a signal and a gas-sensing site: heme-sensing and gas-sensing proteins
Fojtíková, Veronika ; Martínková, Markéta (advisor) ; Hudeček, Jiří (referee)
Heme-containing sensor proteins are heme proteins, which are divided into two groups: heme-sensing and gas-sensing proteins. The function of heme-sensing proteins is affected by heme availability. Association (or dissociation) of heme moiety of heme-sensing protein regulates various physiological functions, including protein kinase activity, transcription and other important functions essential for cell survival. In gas-sensing proteins, heme acts as the sensing site for binding of gaseous molecules (including O2, NO and CO) and indirectly regulates physiological functions, including protein kinase activity, transcription and other important functions essential for cell survival. The recent studies on heme-containing senzor proteins published in scientific journals are summarized in this thesis. The experimental part of this thesis focused on the specific heme-containing sensor protein - a globin-coupled histidine kinase from Anaeromyxobacter sp. strain Fw 109-5 (AfGcHK). The aim of this thesis was to amplified and isolate plasmid carrying gen for AfGcHK. Consequently the protein was expressed in E.coli BL-21(DE3) and the protein was isolated. Based on the results, the isolation process was optimized. Moreover, the purified preparation of isolated AfGcHK was prepared in more than 99% of homogeneity....
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