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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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Study of mechanism of signal transduction in case of two model heme-containing sensor proteins
Mihalčin, Peter ; Martínková, Markéta (advisor) ; Kavan, Daniel (referee)
Heme-based gas sensing proteins belong to a group of proteins that are present in signalling pathways of bacteria. A precise regulation of physiological functions, such as intercellular communication or biofilm production, is essential for the survival of these bacteria and their adaptation to the changing surrounding conditions. Heme-based gas sensors are able to detect the concentration of gas molecules in the local environment via their sensory domain (which contains a heme molecule as the intrinsic detection site) and transmit the signal to the functional domain helping to regulate the adaptation of many processes. These, often pathogenic, processes contribute to extended resistance of bacteria against antibiotics. Heme-based sensors are thus potentially a new therapeutic object of interest in antimicrobial treatment. In order to provide this type of treatment, it is crucial to understand the exact mechanism of intramolecular signal transduction facilitated by heme-based sensors. One of the approaches to unravel these mechanisms is further study of model sensory proteins. This thesis focuses on the analysis of a signal transduction performed by two model globin-coupled heme-based oxygen sensors.
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Molecular mechanisms of signal transduction in model heme-containing oxygen sensor proteins
Stráňava, Martin ; Martínková, Markéta (advisor) ; Obšil, Tomáš (referee) ; Macek, Tomáš (referee)
EN Heme containing gas sensor proteins play important role in bacterial physiology in regulating many processes such as cell differentiation, virulence, biofilm formation or intercellular communication. For their structure, typical modular architecture is characteristic where various sensor domains (usually at the N-terminus) regulate the activity of the catalytic or functional domains (usually at the C-terminus). In this dissertation thesis, we focused on three representatives from the group of oxygen sensing proteins, namely histidine kinase AfGcHK, diguanylate cyclase YddV, phosphodiesterase EcDOS and also on protein RR, which is the interaction partner of AfGcHK. The main aim of the thesis was to study intra-protein/inter-domain signal transduction in two representatives of heme sensor proteins with a globin fold of the sensor domain (AfGcHK, YddV) and in one representative with PAS fold of the sensor domain (EcDOS). Another objective was to describe inter-protein signal transduction in the two component signaling system AfGcHK-RR and structurally characterize these two interacting partners. Emphasis was also placed on the study of the interaction between model sensor domains and different signaling molecules and also on function of individual amino acids involved in the binding of these...
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