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The development of new isotope-coded cross-linkers for label free quantification of proteins
Procházková, Valérie ; Kukačka, Zdeněk (advisor) ; Vaňková, Pavla (referee)
Elucidating the structure of proteins in living organisms is an important step in describing their role. One method of structural biology is chemical cross-linking in combination with mass spectrometry, which is a commonly used tool for characterisation of the tertiary structure of proteins or protein-protein interactions. This method is based on the reaction of a cross-linking agent consisting of two reactive groups linked by an arm of a defined length to form a covalent bond. The main aim of the project was to verify whether chemical cross-linking using istopically labeled cross-linking agents can be used for quantification of two different structural states. The reagents used in this work were DSPU and an isotopically labeled form of DSPUx. Both the DSPU and DSPUx reagents contain a labile urea molecule in their structure, which is cleaved into characteristic fragments during collision-induced dissociation, allowing the identification of cross-linked peptides. Using top-down and bottom-up approaches, it was found on selected proteins (insulin, human carbonic anhydrase, and bovine serum albumin) that DSPU and DSPUx reagents interact with selected peptides and proteins and are suitable for quantification of structural changes. Subsequently, structural differences in the presence (holoform) and...
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Parasite cystatins as inhibitors of cysteine proteases: structural aspects of functional specificity and their evolution
Buša, Michal ; Mareš, Michael (advisor) ; Hudeček, Jiří (referee) ; Kukačka, Zdeněk (referee)
Members of the cystatin family are important inhibitors of cathepsin-type cysteine proteases and are involved in a number of pathologies. Parasite cystatins are attractive target molecules for parasite control, but our knowledge about them is still limited. This work is focused on cystatins of two blood-feeding parasites: the common tick (Ixodes ricinus) as the main vector of Lyme disease and tick-borne encephalitis, and the liver fluke (Fasciola hepatica), the causative agent of fasciolosis. Four novel cystatins were functionally and structurally characterized to determine the structural determinants of their inhibitory specificity and describe them in the context of evolution and physiological role of cystatins. The cystatin FhCyLS-2 from F. hepatica has broad inhibitory specificity and is suggested to play a dual role in the regulation of proteolytic systems in host tissue and the parasite gut. FhCyLS-2 combines the characteristics of two cystatin subfamilies in a unique way and is a model representative of a novel evolutionary group of cystatins identified in several orders of parasitic flukes. Ricistatin and iristatin are salivary cystatins of I. ricinus with immunomodulatory effects on the host caused by an exceptionally narrow inhibitory specificity. It was explained by structural modifications of...
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš ; Schneider, Bohdan (advisor) ; Pichová, Iva (referee) ; Kukačka, Zdeněk (referee)
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Development of chomatography-mass spectrometry technique for separation and identification of glycopeptides applicable in clinical practice
Petroušek, Filip ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Glycoproteins are commonly found in organisms and play an important role in both physiological and pathological conditions. In glycoproteins, different glycans may be present at the same glycosylation site and altered glycosylation of proteins may be a manifestation of disease. As a result, they can be used as markers in diagnostics. For the analysis of glycoproteins, their cleavage into glycopeptides, which are then identified, is often used. Liquid chromatography coupled to mass spectrometry is a widely used method for the study of glycopeptides and hydrophilic interaction chromatography (HILIC) can be used for the separation of glycopeptides. This type of chromatography allows separation of glycopeptides based on the properties of both the peptide and the bound glycan. The first aim of this work was to determine the extent to which the charge of the peptide backbone of glycopeptides affects their separation by HILIC. Penta-HILIC and HILIC-B columns were used for this purpose. Penta-HILIC contains a polyhydroxyl stationary phase, while the stationary phase of HILIC-B consists of silica gel modified with positively charged aminopropyl groups. IgG1 and IgG2 glycopeptides were separated on both columns. To determine the extent of the effect of charge and hydrophobicity during separation on HILIC-B,...
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The development of new tyrosin binding cross-linkers for structural characterization of proteins
Karpíšek, Michael ; Kukačka, Zdeněk (advisor) ; Talacko, Pavel (referee)
Proteins are cornerstones of all living organisms. A lot of energy is invested in studying structures of proteins, because their function is determined by their structure. One of the techniques used to access the structure of proteins is chemical cross-linking in combination with mass spectrometry. In spite of their number, most of the cross-linkers bind few aminoacids such as lysine, cysteine or glutamic and aspartic acid. In our work we tried to develop tyrosin-binding cross-linkers. Using top down and bottom up approaches we studied the influence of N-hydroxysuccinimide esters and imidazole on the reactivity of the cross-linker in different pH on model proteins. According to the acquired data there is a difference in overal reactivity and specifity between the used cross-linkers. The cross-linkers containing imidazole modified tyrosine more although the difference was small and binding of tyrosine was not selective. [IN CZECH]
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The use of protein radical footprinting by Togni reagents in structural biology
Fojtík, Lukáš ; Kukačka, Zdeněk (advisor) ; Kolenko, Petr (referee)
Structural proteomic methods such as an ion mobility mass spectrometry, chemical cross- linking or covalent labeling have been established as powerful tools for structural studies of biomolecules in general. These methods have significantly contributed to the expansion of our knowledge about biomolecular functions, their dynamics and molecular interactions and therefore led to the understanding of important biological processes occurring in a cell. We decided to spread these methods and we developed a new radical labeling technique relaying on Fluor-alkyl radicals that does not require a laser dissociation pf hydrogen peroxide. We exploited the potential of Togni reagents to form Fluor-alkyl radicals by reducing agent under native conditions. The induction of Fluor-alkyl radicals was triggered by ascorbic acid and the labeling pulse was stopped by tryptophan. The modified proteins were analyzed by top down or bottom up approach using high resolution mass spectrometry. In case of top down approach, several fragmentation techniques (CID-ECD, ETD) were tested for protein analysis while in case of bottom up approach the analyzed proteins were digested by trypsin and separated on reverse phase column online coupled to mass spectrometer. As the model biomolecules we chose a 20 proteinogenic amino acids...
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Characterization of protein structures using chemical cross-linking and mass spectrometry.
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee) ; Hernychová, Lenka (referee)
Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.
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Preparation of cytochrome b5 mutants containing photoreactive amino acids, and their crosslinking with the interaction partners
Landl, David ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Cytochrome b5 is an electron transport protein of a clinically prominent mixed-function oxygenase (MFO) system. This system participates in the first stage of xenobiotic biotransformation. The hydrofility of xenobiotics is increased by introduction of an oxygen atom into their structure. The MFO system also activates or deactivates certain drugs and carcinogens. Cytochrome b5 affects reactions catalyzed by the terminal oxygenases of the system - cytochromes P450. Electrons are donated to cytochrome b5 by redox partners NADH:cytochrome b5 reductase and NADPH:cytochrome P450 reductase. The aim of this work was to demonstrate capability of photo-crosslinking approach to fixate transient interactions within MFO system. Covalent complexes obtained by this technique could be further analyzed by mass spectrometry, which can provide structural information about the binding sites of the proteins. We prepared a mutant cytochrome b5 comprising photo-reactive methionine analogue in the position 41 of the sequence. We expressed the protein employing E. coli B834 (DE3) auxotrophic strain in 300 ml of the limit medium supplemented with L-2- amino-5,5-azi-hexanoic acid (photo-methionine). The rate of the unnatural amino acid incorporation was determined by mass spectrometry and it was about 40 % after 16 hours of...
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Preparation of transcription factor FOXK2 - DNA binding domain
Kobrle, Lukáš ; Kukačka, Zdeněk (advisor) ; Bělonožníková, Kateřina (referee)
Transcription factors are specific proteins, which are able to regulate transcription and which are divided into individual families based on structural motifs. One of these families is the FOX family, which also includes the transcription factor FOXK2, which was the studying subject of this work. The FOXK2 protein regulates many cellular processes, including cell proliferation and metabolism and plays an important role in carcinogenesis, as its high expression has been reported in cases of lung, liver and kidney cancer. In this work, the DNA binding domain of the transcription factor FOXK2 was prepared by recombinant expression of Escherichia coli cells. The obtained protein was afterwards purified and its interaction with DNA was studied by native mass spectrometry.
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