Search

ID: 1125 In: Physical chemistry chemical physics, Vol. 17, no. 26 ( 2015), p.17007-17015. Summary: AbstractWe present time-resolved step-scan FTIR evidence for the role of the distal Y43 and L65 residues in controlling the ligand dynamics in the signal transducer protein YddV.

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...

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]

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...

Tumor suppressor p53 is one of the most studied proteins in terms of cancer and the mechanism of its formation. The general function of p53 is based on the transcriptional regulation of various genes, which can differently influence numerous cellular processes. Recent studies revealed a relationship between p53 and iron homeostasis within the cell. In particular, p53 was shown to interact with a molecule of heme, and this interaction ultimately disrupts the DNA-binding ability of p53 and promotes its proteasomal degra- dation. This work focuses on a detailed description of heme binding to the p53. For this purpose, we isolated two forms of p53, heme-free and heme-bound. We discovered that conformational dynamics of heme-free and heme-bound p53 differ, with the latter exhibi- ting a higher degree of flexibility. We also confirmed previous reports that heme indeed interacts with a cysteine residue in a specific manner. However, heme binding does not disrupt the oligomeric state of p53 or its native zinc binding ability. Finally, we showed that heme-bound p53 exhibits severely impaired DNA-binding ability as opposed to the heme-free form. Keywords: heme, sensor proteins, p53 protein, transcription factor, intrinsically disor- dered proteins

DNA amplification is an important tool in diagnosis of infectious diseases. The most commonly used method in laboratories is the polymerase chain reaction, which, does not meet the need for a fast and inexpensive method. It was therefore necessary to develop a method that would be sensitive, fast, specific and accessible, for example, in developing countries with limited access to instrumentation. It appears that LAMP could be this method. Unfortunately, the LAMP method encounters considerable false positivity, which must be prevented in order to be used in practice. The subject of the presented bachelor thesis is the introduction of a unique LAMP method to detect the presence of DNA Streptococcus pneumoniae and the study of the use of uracil- DNA glycosylase to prevent contamination of samples with DNA formed in previous amplification reactions. The LAMP method of DNA detection Streptococcus pneumoniae was successfully introduced, then the method was optimized even in the presence of deoxyuridine triphosphate. After determining the appropriate deoxyuridine triphosphate concentration for LAMP amplification, the resulting Streptococcus pneumoniae DNA amplification products were treated with uracil-DNA glycosylase. This procedure was shown to have the potential to prevent false positivity of other...

Bacterium Streptococcus pneumoniae is currently one of the most serious pathogenic microorganisms posing a threat to children under the age of 5, especially in developing countries. A suitable detection method is a tool in the fight against this pathogenic microorganism. The presented bachelor thesis deals with the comparison of molecular- biological methods of detection of the bacterium Streptococcus pneumoniae, specifically by quantitative polymerase chain reaction and loop mediated isothermal amplification of DNA. A standard protocol for the detection of Streptococcus pneumoniae using the "golden standard" of molecular diagnostics, that is the quantitative polymerase chain reaction, has been established. An alternative to quantitative polymerase chain reaction detection has been the practical laboratory introduction of a loop mediated isothermal DNA amplification method using published "primers". In order to expand the diagnostic window of the loop mediated isothermal amplification method, and to combat the false positive of the negative control, a set of new "primers" for the detection of Streptococcus pneumoniae was designed and tested. The result of the work was a new, rapid and reliable protocol for the detection of Streptococcus pneumoniae using a combination of newly designed and published...

Haemophilus influenzae is one of the main initiators of meningitis and pneumonia in children. Implementation of fast, cheap and instrumentally accessible method for detection of this pathogen would enable an early and targeted treatment of patients. The development of amplification methods in the last decades enables, apart from commonly used PCR method, application of alternative approaches, such as the LAMP. The focus of this Bachelor thesis was the study (research) of the alternative method LAMP as a tool for detection of Haemophilus influenzae. The LAMP method was successfully implemented for Haemophilus influenzae, however, it has contended with the false positive results of negative control in case of longer incubation times. Therefore, the optimized LAMP method was designed in presence of deoxyuridine triphosphate and uracil-DNA glycosylase. Its aim was to change the structure of LAMP products via the incorporation of uracils to amplified regions of DNA and subsequent removal of uracils with influence of uracil-DNA glycosylase, and therefore prevent their replication during potential contamination of reaction mixtures and consequently reduce the risk of false positive results of negative controls to minimum. The concentration of deoxyuridine triphosphate in reaction mixtures was optimized...

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

Heme sensor proteins perform a variety of important functions in both prokaryotic and eukaryotic organisms. Heme-regulated inhibitor (HRI) is an example of a eukaryotic heme-sensor protein, which catalyzes the phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α). In this bachelor thesis, the pET-21c(+)/eIF2α plasmid was amplified and its authenticity for the eIF2α expression was verified with the use of two independent methods. Next, HRI and eIF2α were produced using the recombinant expression in E. coli BL-21(DE3) cells transformed with the pET- 21c(+)/eIF2α and pET-21c(+)/HRI plasmid, respectively. Both proteins were then isolated from the cells and purified with the use of affinity chromatography and gel permeation chromatography. eIF2α was obtained in sufficient yield (560 μg out of 1 l of TB medium) and purity (90%). A lower yield (25 μg out of 1 l of TB medium) and purity (20%) was reached in the case of HRI. On the other hand, the authenticity of the HRI product was confirmed using spectrophotometric characterization and its enzyme activity was verified as well. Pilot experiments showed that GTP may replace ATP in the process of eIF2α phosphorylation, while UTP and CTP may not.

Xenobiotics, such as pharmaceuticals, food additives, environmental pollutants, and dietary bioactive compounds in organism are metabolized by various enzymes, resulting in their bioactivation or detoxification. Identification of structure of resulting metabolites is important for their detection in bodily fluids and tissues for diagnostic and forensic purposes. This thesis reviews known biochemical processes and enzymes involved in xenobiotic metabolism, including cytochromes 450 (CYP) and flavine monooxygenases (FMO). Given that biochemical reactions are to a major extent composed of electron-transfer reactions (i.e. oxidation and reduction), the thesis includes a section dealing with the practical approaches to determination of the oxidative or reductive mechanism of bioactive compounds.

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...