|
|
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...
|
|
| |
|
| |
|
|
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...
|
|
|
The influence of stochastic behaviour of ion channels on the signal and information transfer at excitable neuronal membranes
Šejnová, Gabriela ; Kuriščák, Eduard (advisor) ; Maršálek, Petr (referee)
The stochastic behavior of voltage-gated ion channels causes fluctuations of conductances and voltages across neuronal membranes, contributing to the neuronal noise which is ubiquitous in the nervous system. While this phenomenon can be observed also on other parts of the neuron, here we concentrated on the axon and the way the channel noise influences axonal input-output characteristics. This was analysed by working with our newly created computational compartmental model, programmed in Matlab environment, built up using the Hodgkin-Huxley mathematical formalism and channel noise implemented via extended Markov Chain Monte Carlo method. The model was thoroughly verified to simulate plausibly a mammalian axon of CA3 neuron. Based on our simulations, we confirmed quantitatively the findings that the channel noise is the most prominent on membranes with smaller number of Na+ and K+ channels and that it majorly increases the variability of travel times of action potentials (APs) along axons, decreasing thereby the temporal precision of APs. The simulations analysing the effect of axonal demyelination and axonal diameter correlated well with other finding referred in Literature. We further focused on spike pattern and how is its propagation influenced by inter-spike intervals (ISI). We found, that APs fired...
|
|
|
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...
|
|
|
β-Arrestin and its role in signal transduction
Marková, Vendula ; Novotný, Jiří (advisor) ; Nerandžič, Vladimír (referee)
β-Arrestin is a ubiquitous protein in cells, where it is involved in signal transduction and can affect different cellular processes. β-Arrestin cooperates with G protein-coupled receptors (GPCRs). Binding of β-arrestin to a receptor after its activation by a relevant ligand results in attenuation of signal transduction through the cognate G proteins, the process called desensitization, which can be associated with receptor intrenalization. Besides that, β-arrestin acts as adaptor for different molecules, which participate in signal transduction. β-Arrestin also has a role in a regulation of transcription in the cell nucleus. Finally, β-arrestin is explored in research focused on the development of a new type of drugs, so called biased ligands. After binding to a GPCR, these ligands can initiated only one specific activity of the receptor and affect relevant signaling cascades. Powered by TCPDF (www.tcpdf.org)
|
|
|
Activation of the initiation caspases and regulation of their activity
Votavová, Barbora ; Anděra, Ladislav (advisor) ; Černý, Jan (referee)
Caspases are the key proteins participating in both activation and execution of apoptosis. Extrinsic or intrinsic apoptotic signaling leads to sequential activation of the initiation and execution caspases. Activation of initiator caspases is mediated by their processing in multiprotein complexes and activated initiator caspases then specifically cleave and thus activate the effector caspases. These then cleave a number of structural and functional proteins, which consequently leads to cellular selfdestruction and its breakdown to apoptotic bodies. Considering the fundamental significance of the initiation of apoptosis, the activation as well as the activity of initiator (but also effector) caspases is strictly regulated at several levels. Primary the intensity and character of the recieved signal is crucial for the effective formation of the caspase activation complex. Then the concentration of intracellular ionts, nucleotides and various proteins (proteins from the Bcl-2 family, inhibitors of apoptosis (IAPs), heat shock proteins,…) can also highly influence individual steps of caspase activation. Caspases themself can be posttranslationally modified (phosphorylated, ubiquitylated,…) and their activity can be either suppressed or also enhanced. All these processes form complex regulatory network serving...
|
|
| |
|
| |
guest ::
