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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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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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Preparation of proteases for protein structure studies.
Jirečková, Barbora ; Man, Petr (advisor) ; Vaňková, Pavla (referee)
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is a method allowing the study of protein structure and dynamics. Its spatial resolution is given by the proteolysis step that is included in the HDX-MS workflow. Most widely used pepsin has however some limitations and use of a single protease often does not provide optimal spatial resolution. Several publications have emphasized the importance of the alternative proteases nepenthesin-2 (Nep-2) and aspergillopepsin (XIII) cleaving, in contrast to pepsin, after basic amino acids. In studies targeting proline-rich proteins, another enzyme, prolyl endoprotease from Aspergillus niger (AnPEP), is gaining importance. This work focuses on the characterization of immobilized AnPEP in combination with pepsin, aspergillopepsin or Nep-2 for their application in HDX-MS. First, columns with only one protease were tested on a set of model proteins. It was found that immobilized AnPEP did not have optimal cleavage characteristics compared to the other proteases. In order to combine the advantages of the proteases mentioned above, the model proteins were digested using columns with AnPEP coimmobilized with pepsin, Nep-2 or XIII and also using two protease columns in series (always AnPEP column with pepsin, Nep-2 or XIII column in both...
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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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Adaptive evolution of Toll-like receptors in birds
Velová, Hana ; Vinkler, Michal (advisor) ; Elleder, Daniel (referee) ; Novák, Karel (referee)
Adaptive evolution of Toll-like receptors in birds Hana Velová, PhD thesis 6 Abstract Toll-like receptors (TLRs) are one of the key and presumably also evolutionary most original components of animal immune system. As Pattern recognition receptors they form the first line of innate immune defence against various pathogens. The proper receptor binding of pathogenic ligands is crucial for their correct recognition and for subsequent triggering of an appropriate immune response. Because there exists a direct interaction between the receptor surface and the pathogenic ligand, host-pathogen coevolution on molecular level can be predicted. Thus, through variability of their ligands, TLRs are exposed to extensive selective pressures that may be detected on both genetic and protein levels. Surprisingly, the variability we revealed in birds is even higher than previously expected based on the reports from other vertebrates, mainly mammals. In my doctoral thesis I summarise the results of my contribution to the avian TLR research. We were the first who experimentally verify the absence of functional TLR5 in several avian species and duplication of TLR7 in others. We finally resolved the origin of duplication in TLR1 and in TLR2 family. An important part of my research project focused on the prediction of potentially...
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Characterization of proteins of 2'-5' oligoadenylate pathway by means of vibrational spectroscopy
Víšová, Ivana ; Kopecký, Vladimír (advisor) ; Bednárová, Lucie (referee)
The work concerns to structural characterization of two important proteins of 2'-5' oligoadenylate pathway participating in an immune response of organism to a viral infection. Studied proteins were ankyrin domain of mouse RNase L, the C-terminal part of human phosphodiesterase 12 and the complete human phosphodiesterase 12. The proteins were characterized by Raman spectroscopy, infrared spectroscopy, electronic circular dichroism, dynamic light scattering and in addition by two non-spectroscopic methods- differential calorimetry and electrophoresis. For each protein the secondary structures, thermal stability, weight of oligomers and generally a basic characterization by above mentioned methods were provided.
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Analysis of the mechanism of action of metallacarborane inhibitors of HIV PR
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...
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Conformational Behaviour of Small Peptide Fragments Studied by the Quantum Chemical Methods
Kalvoda, Tadeáš ; Rulíšek, Lubomír (advisor) ; Vondrášek, Jiří (referee)
To what extent conformational preference of short peptide sequences within proteins determine their three-dimensional structure? Large-scale quantum chemical calculations coupled with modern solvation methods represent unique set of tools to elucidate key determinants of the biomolecular structure ab initio. Full conformational sampling was performed on model systems representing short peptide fragments. The computed data reveal some of the underlying physico-chemical principles determining the spatial structure of proteins, and provide very important data for finding and tuning the optimal algorithm that may provide a full coverage of (ideally all) low-energy conformers. Keywords: Conformational space, peptide fragments, protein structure, solvation methods, Ramachandran plot, DFT-D3 methods
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Adaptive evolution of Toll-like receptors in birds
Velová, Hana ; Vinkler, Michal (advisor) ; Elleder, Daniel (referee) ; Novák, Karel (referee)
Adaptive evolution of Toll-like receptors in birds Hana Velová, PhD thesis 6 Abstract Toll-like receptors (TLRs) are one of the key and presumably also evolutionary most original components of animal immune system. As Pattern recognition receptors they form the first line of innate immune defence against various pathogens. The proper receptor binding of pathogenic ligands is crucial for their correct recognition and for subsequent triggering of an appropriate immune response. Because there exists a direct interaction between the receptor surface and the pathogenic ligand, host-pathogen coevolution on molecular level can be predicted. Thus, through variability of their ligands, TLRs are exposed to extensive selective pressures that may be detected on both genetic and protein levels. Surprisingly, the variability we revealed in birds is even higher than previously expected based on the reports from other vertebrates, mainly mammals. In my doctoral thesis I summarise the results of my contribution to the avian TLR research. We were the first who experimentally verify the absence of functional TLR5 in several avian species and duplication of TLR7 in others. We finally resolved the origin of duplication in TLR1 and in TLR2 family. An important part of my research project focused on the prediction of potentially...
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Comparative structural analysis of Borreliella spp. virulent factors focusing on their surface topology conservation
Zdrha, Alois ; Drbal, Karel (advisor) ; Nunvář, Jaroslav (referee)
Gram-negative bacteria of genus Borreliella cause the nowadays spreading illness Lyme borreliosis. However, their classification as gram-negative bacteria is rather mislea- ding because they differ in their complex genome, pathogenic adaptation to their hosts and composition of their outer membrane. One species of borreliella can harbour up to 23 variable plasmids and one conserved chromosome and they still lack many crucial proteins needed for synthesis of key compounds. Hence, they have to obtain these compounds from the host. However, in order for borreliella to utilise the metabolic processes of the host, it has to first survive its immune response. Therefore borreliella contains many virulent factors including highly variable surface lipoproteins. The variability is a major obstacle to overcome when using their surface epitopes for detection and vaccine development because most of the dominant antigenic epitopes of borreliella are usually parts of the most variable regions of the lipoproteins. Nowadays, we can use many different algorithms which determine evolutionary conserved epitopes based on analysis of sequences of given lipoprotein, in order to find suitable targets for antibodies. In general, conserved epitopes are more suitable for vaccines, whereas more variable epitopes are better...
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