National Repository of Grey Literature 14 records found  1 - 10next  jump to record: Search took 0.00 seconds. 
The two-component signal transduction system in bacteria utilizing heme analogues
Bečková, Alexandra ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
6 Abstract Hemoproteins are essential components of both prokaryotic and eukaryotic organisms, where they play a role in fulfilling many significant life functions. According to their function, hemoproteins can be divided into several types. One specific type of hemoproteins, which was also the object of interest in this bachelor thesis, is the heme sensor proteins. The function of part of these sensor hemoproteins is the ability to detect diatomic gas molecules and subsequently bind them to the heme molecule in the protein. This process leads to changing the properties of the sensor hemoprotein and the signal then becomes part of the signalling pathways. An example of sensor hemoprotein is the histidine kinase, with a globin structure of the sensor domain, from Anaeromyxobacter sp. Fw 109 (AfGcHK), which was studied during the experimental part of the bachelor thesis. First, a transformation of E. coli BL-21 (DE3) cells with the pET21c(+)/AfGcHK plasmid was performed, followed by cell cultivation and expression of the AfGcHK protein in the cells. The expressed AfGcHK protein was isolated in two forms. One form represented AfGcHK protein containing heme, that is, with iron incorporated in its structure. The second form represented AfGcHK proteins containing a heme analogue, where nickel was incorporated...
Heme sensor proteins as potential biomarkers of cellular and oxidative stress processes induced by ionizing radiation
Vávra, Jakub ; Martínková, Markéta (advisor) ; Souček, Pavel (referee) ; Tichý, Aleš (referee)
[IN CZECH] Ionizing radiation is a potential inducer of the oxidative stress processes in cells. As a result, reactive radicals are formed in the intracellular space modifying the essential biomolecules. Ionizing radiation has either direct (radiation sickness) or indirect (malignant processes) effect on the organism. Therefore, a fast determination of the dose is required when suspected irradiation of the organism occurs. However, a method routinely applicable, fast enough and at the same time suitable for dose estimation based on biomarkers has not been developed so far. The aim of this thesis is to describe new properties of the selected heme sensor proteins and discuss their potential importance for the cellular adaptation to oxidative stress conditions. Specifically, the thesis is focused on two eukaryotic proteins, heme regulated inhibitor (HRI) and transcription factor p53. The study of functional regulation as well as the conformational changes of these proteins induced by heme is greatly emphasized. Besides, the optimization of the key experimental methods was conducted. Specifically, Phos-tag electrophoresis was applied for the kinetics study of HRI wild type and its Gly202Ser mutant form, which is a characteristics of lung cancer development. Unsurprisingly, for both HRI forms studied,...
Detail enzymatic characterization of a model heme-containing oxygen sensor
Vojáčková, Lukrécie Sophie ; Martínková, Markéta (advisor) ; Čermáková, Michaela (referee)
This thesis focuses on heme-based gas sensors, particularly phosphodiesterase from the bacterium Escherichia coli, referred to as EcDOS. The enzyme catalyzes the degradation of c-di-GMP, an important signaling molecule in bacteria that affects cellular processes, such as bacterial motility or biofilm formation. The thesis deals with detailed enzyme kinetics of protein forms in different redox and ligand states of the heme iron ion [Fe(III), Fe(II) and Fe(II)-O2], as well as a mutant form of the enzyme (EcDOS H77A) which does not bind heme. Results confirmed that the EcDOS WT Fe(II)-O2 form has higher kcat values than the EcDOS form with the ferrous ion of heme in the reduced state. Other significant result was that the enzyme activity is affected not only by the state of the heme iron ion but also by the presence and concentrations of divalent metal cations. The presence of the metal cation is essential for enzyme function, and suitable metal ions that stimulate enzyme activity are Mg2+ , Mn2+ and Zn2+ or their mixtures, which act synergistically on enzyme activity under chosen conditions. Analysis by ICP-MS also showed that Zn2+ cations are natural components of the enzyme. Thus, for further kinetic studies, it would be appropriate to use Zn2+ or mixtures of metal ions that are physiological for...
Structure and function relationships of model hemoproteins
Lengálová, Alžběta ; Martínková, Markéta (advisor) ; Hudeček, Jiří (referee) ; Muchová, Lucie (referee)
Heme is one of the most important and most studied cofactors that are essential for proper function of many proteins. Heme-containing proteins comprise of a large group of biologically important molecules that are involved in many physiological processes. The presented dissertation is focused on two groups of heme sensor proteins, namely prokaryotic heme-based gas sensors and eukaryotic heme-responsive sensors. Heme-based gas sensors play an important role in regulation of many bacterial processes and consist usually of two domains, a sensor domain and a functional domain. The dissertation thesis aims at the study of two model bacterial heme-based gas sensors, histidine kinase AfGcHK and diguanylate cyclase YddV, in order to elucidate their mechanism of interdomain signal transduction. Using X-ray crystallography and hydrogen-deuterium exchange coupled to mass spectrometry approaches, significant differences in the structure of the AfGcHK protein between the active and inactive forms were described. The signal detection by the AfGcHK sensor domain affects the structural properties of the protein, and these conformational changes then have indirect impact on the enzyme activity of the functional domain. Further, the dissertation pays more attention to the effect of a sensor domain dimerization...
Study of the effect of heme analogues on the structural-functional characteristics of a model representative of heme sensor proteins
Ďatko, Peter ; Martínková, Markéta (advisor) ; Vávra, Jakub (referee)
Heme sensor proteins allow bacteria to react to changes of concentration of certain molecules in their environment. This reaction depends on the coordination of the ligand to the heme iron atom. Model representative of this signaling system is a histidine kinase containing a sensor domain with a globin structure, AfGcHK. The aim of this bachelor thesis was to prepare and characterize a modified form of AfGcHK containing manganese within its protoporphyrine complex. To express the protein, E. coli BL-21 (DE3) cells were transformed using a plasmid pET21c(+)/AfGcHK. The protein was isolated and purified using affinity chromatography and gel chromatography. To determine its enyzmatic activity, polyacrylamid gel electrophoresis in the presence of sodium dodecyl sulfate with Phos-Tag was used. It was determined, that this novel form of AfGcHK is enzymatically active. Spectroscopic analysis has shown, that the modified form of AfGcHK containing manganese within its protoporphyrine complex is susceptible to reduction by sodium dithionate. Key words: heme, heme sensor proteins, oxygen sensors, signal transduction [IN CZECH]
Biochemical characterization of a model heme sensor protein containing a heme analog
Hlubučková, Darina ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
An integral part of the life of cells is cell signaling, which is ensured, among other things, by heme proteins, specifically by their members called heme sensor proteins. Heme sensor proteins are divided into two groups, heme sensor proteins detecting heme, which are found more in eukaryotic cells, and heme sensor proteins detecting gaseous molecules, which are more typical of prokaryotic cells. The gas molecules in this case can be CO, NO and O2. A family of oxygen-detecting heme sensor proteins is crucial for bacteria that must adapt to changing oxygen concentrations in the environment. One of these bacteria is the soil bacterium Anaeromyxobacter sp., strain Fw 109-5, which contains a heme sensor protein with the globin structure of the sensor domain and the histidine kinase activity of the functional domain (AfGcHK), enabling the bacteria, among other things, to form a biofilm. As part of the theoretical part of this thesis, the current knowledge about heme sensor proteins was summarized, with a focus on the AfGcHK protein. In the practical part of this bachelor's thesis, bacterial cells E. coli BL-21 (DE3) were transformed with the plasmid pET21(+)/AfGcHK containing the gene encoding AfGcHK, from which two forms of AfGcHK were subsequently isolated, the natural form and the form containing a...
Structural characterization of a model heme-containing oxygen sensor
Tajovská, Eva ; Martínková, Markéta (advisor) ; Ryšlavá, Helena (referee)
One subgroup of hemoproteins are heme-based gas sensors, which are able to detect biatomic gas molecules in their immediate surroundings. Upon binding of a gas molecule to the heme iron in a sensor domain of these proteins or, conversely, upon its dissociation from the heme iron, the signal is then transmitted from the sensor domain to a functional domain and subsequent regulation of important cellular functions occurs. Understanding the regulatory mechanism of gas sensors is key to potentially manipulating their function. Such knowledge would then allow the use of heme-based gas sensors as therapeutic targets for the development of next-generation antibiotics, if we take into account their presence in pathological bacteria. The diploma thesis focuses on a model heme-based gas sensor, the oxygen sensor EcDOS from E. coli, and its apoform, EcDOS His77Ala. Both proteins were prepared by recombinant expression and purification, and subsequently spectrophotometrically characterized. Using gel permeation chromatography, the oligomeric states of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS His77Ala were determined under different conditions (different temperatures of protein incubation, presence of c-di-GMP substrate etc.). Furthermore, the structural dynamics of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS...
Heme sensor proteins sensing both heme and CO
Andrlová, Dominika ; Martínková, Markéta (advisor) ; Libus, Jiří (referee)
Heme, a protoporhyrin IX iron complex, is an important component of many proteins necessary for oxygen transfer, storage and activation, as well as for electron transfer. Another group of hemoproteins includes heme sensor proteins. They are either capable of detecting heme itself, which can regulate in turn the sensor function (heme-responsive sensors) or heme forms a binding site for small gas molecules (O2, CO and NO) and the heme-based gas sensors are regulated by these diatomic gases. However, in the case of some proteins their classification is not clear showing a properties of both heme sensor proteins families. Their functions are regulated by heme interaction and a further change in their function after binding of a gas molecule to heme was observed. This summary search is focused on specific representatives of heme-responsive sensors (which function is regulated by heme binding), in which the further influence of the CO molecule on their functions have recently been observed. It is discussed whether some heme-responsive sensors are also heme-based CO sensors aiming the most recent findings about the selected specific heme sensors representatives. Key words: heme, heme sensor proteins, heme-based gas sensors, CO sensors, heme-responsive sensors, heme redox sensors
Structure and function relationships of model hemoproteins
Lengálová, Alžběta ; Martínková, Markéta (advisor) ; Hudeček, Jiří (referee) ; Muchová, Lucie (referee)
Heme is one of the most important and most studied cofactors that are essential for proper function of many proteins. Heme-containing proteins comprise of a large group of biologically important molecules that are involved in many physiological processes. The presented dissertation is focused on two groups of heme sensor proteins, namely prokaryotic heme-based gas sensors and eukaryotic heme-responsive sensors. Heme-based gas sensors play an important role in regulation of many bacterial processes and consist usually of two domains, a sensor domain and a functional domain. The dissertation thesis aims at the study of two model bacterial heme-based gas sensors, histidine kinase AfGcHK and diguanylate cyclase YddV, in order to elucidate their mechanism of interdomain signal transduction. Using X-ray crystallography and hydrogen-deuterium exchange coupled to mass spectrometry approaches, significant differences in the structure of the AfGcHK protein between the active and inactive forms were described. The signal detection by the AfGcHK sensor domain affects the structural properties of the protein, and these conformational changes then have indirect impact on the enzyme activity of the functional domain. Further, the dissertation pays more attention to the effect of a sensor domain dimerization...

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