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Protein Domains Prediction
Valenta, Martin ; Martínek, Tomáš (referee) ; Burgetová, Ivana (advisor)
The work is focused on the area of the proteins and their domains. It also briefly describes gathering methods of the protein´s structure at the various levels of the hierarchy. This is followed by examining of existing tools for protein´s domains prediction and databases consisting of domain´s information. In the next part of the work selected representatives of prediction methods are introduced. These methods work with the information about the internal structure of the molecule or the amino acid sequence. The appropriate chapter outlines applied procedure of domains´ boundaries prediction. The prediction is derived from the primary structure of the protein, using a neural network The implemented procedure and its possibility of further development in the related thesis are introduced at the conclusion of this work.
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Structure and interaction of human 14-3-3 regulatory protein using in vitro photoaffinity labelling in combination of protein nano-probes and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Dračínská, Helena (referee)
This thesis is focused on the study of the structure and mechanism of human 14- 3-3 protein, which is one of the important regulatory proteins present in all eukaryotic cells. Nowadays it is known seven isoforms of this protein in mammals. Although their crystal structure shows a high similarity, their mutual comparison reveals some changes. The aim of this work is to prepare experimental tools for verification whether the differences in the crystal structure of the ζ isoform are present in solution and how the structure-functional mechanism of this isoform is affected. The otimization of 14-3- 3zeta recombinant protein expression with incorporated a photo-labile analog of leucine in the protein sequence was performed using limiting medium with prokaryotic expression system of E. coli BL-21 DE3 Gold or system of auxotrophic E. coli K-12 with non-functional leucine biosynthesis.
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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
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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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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Computer simulations of protein structures using coarse-grained models
Halda, Miloš ; Nová, Lucie (advisor) ; Limpouchová, Zuzana (referee)
The main part of this bachelor's thesis is an operational software for coarse- grained protein simulations, suitable for testing of new potential functions. The program is using a pivot-based proposal of new states and Monte Carlo evaluation of the proposed state. The program also allows to use a simulated annealing technique with the linear temperature decrease. Mean estimation and error estimation using the Block Method are implemented for evaluation of the simulations. The software was tested on a several proteins. The simulations provided expected results for shorter chains (88 and less aminoacids), but simulations of longer chains (111 and more aminoacids) have shown the software limitations. Several options for the software improvement regarding the new state proposal for simulations of longer chains were discussed. 1
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Modulation of interactions of cytokines and their receptors
Kolářová, Lucie ; Schneider, Bohdan (advisor) ; Rozbeský, Daniel (referee) ; Osička, Radim (referee)
Protein-protein interactions and interactions with other molecules including DNA and RNA, play an important role in a range of biological activities and processes in all living cells. Understanding of protein-protein interactions, new approaches, and tools for their modulations are valuable for medicine, biotechnology, and drug development. We used the interleukin-10 family of cytokines as a model system for our research of biological interactions and modulation of their functions. A key prerequisite to study biological processes and a detailed understanding of biomolecular interactions is a recombinant protein that is stable under a broad range of conditions. Recombinant protein expression in sufficient yield and quality is often a challenging task. Therefore, we aimed at developing new approaches in protein design and production. In the first part of our study, we modified IL-24, a member of the IL-10 family to increase its expression and stability. We demonstrated that protein engineering is a powerful tool in research of difficult protein targets. In the second part of our study, we adopted new approaches in designing new protein scaffolds suitable for use in the ribosome and yeast display techniques. Protein scaffolds have become promising alternatives to antibodies in protein drug...
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LacZ-alpha complementation peptide as a tool for molecular evolution studies
Ptáčková, Barbora ; Hlouchová, Klára (advisor) ; Vaněk, Ondřej (referee)
Proteins are the key structural and functional molecules of living organisms. Although the last decades have brought a lot of knowledge about their structural and functional characteristics, science still lacks very basic answers about how these properties evolved. Current predominant opinions suggest that early genetic code contained only a subset of today's canonical amino acids. Both exogenous and endogenous sources of prebiotic amino acids imply that even though the prebiotic amino acid repertoire was very broad, only about half of the proteinogenic amino acids were present. It follows that the ''evolutionary new'' amino acids were added to the genetic coding system only after the evolution of their biosynthetic pathways. From the current scientific knowledge it is unclear whether proteins composed of "evolutionary old" amino acids could serve basic metabolic functions and if today's proteins could be "reversely-evolved" to be composed of only such a subset of amino acids while maintaining their structural and functional integrity. These questions lie at the core of this study. This thesis aims to test a starting methodology that would randomize "new" amino acid positions by "old" amino acids in the sequence of LacZ-alpha peptide. This peptide was selected as a target model protein because it...
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Therapeutic use of alternative protein binders targeting tumor biomarkers in clinical testing of oncology patients
Tauš, Petr ; Drbal, Karel (advisor) ; Lepšík, Martin (referee)
Almost until the end of the last century, antibodies (aka immunoglobulins) were considered the only class of specific binding proteins. The discovery of hybridoma technology in 1975 had enabled the production of monoclonal antibodies and after twenty years some of them have entered clinical practice. Meanwhile, the first non-immunoglobulin protein scaffold, in which new specific binding sites could be introduced was discovered. To date, many different alternative scaffolds have been described, but only a few of them are being further developed for diagnostics, therapeutics or tools in basic research. Since these structures are overcoming the drawbacks of immunoglobulin structure, which are big size, expensive production and difficult rational design, they have potential to replace and exceed them. In this bachelor's thesis all the alternative scaffolds in development are summarized. Moreover, their advancements in clinical trials are described and compared with approved therapeutics based on immunoglobulin structure.
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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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