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Biochemical characterization of a model heme sensor protein
Tajovská, Eva ; Martínková, Markéta (advisor) ; Jeřábek, Petr (referee)
Hemoproteins play a lot of important roles within the living organism. One of these being the sensor function - heme sensor proteins are able to detect the changing concentration of heme in organisms. Heme itself serves as a signalling molecule for these proteins. Heme-based gas sensor proteins use a biatomic gas molecule as a signalling molecule. Signal transduction begins once the gas molecule is bound to the heme molecule which is already tightly bound in the protein sensing domain. Both these types of signalling regulate a number of physiological processes in the prokaryotic and eukaryotic organisms. The theoretical part of the bachelor thesis summarizes recent scientific studies on heme sensor proteins, while the experimental part focuses on the properties of a specific model sensor hemoprotein. A direct oxygen sensor from E. coli (EcDOS) was selected as a model protein. Furthermore, the thesis deals with a truncated form of the EcDOS protein, an isolated sensor domain with a PAS structure (EcDOS-PAS). The experimental part aimed at expressing and isolating of both mentioned proteins from E. coli BL-21 (DE3) cells. Finally, both the EcDOS and EcDOS-PAS proteins were preliminarily characterised and their properties compared. The yield of the EcDOS-PAS was several times higher than the yield of...
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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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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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