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Bioinformatics Tool for Protein Structure Prediction
Plaga, Michal ; Burgetová, Ivana (referee) ; Martínek, Tomáš (advisor)
The goal of this thesis is test and comparation of the offline tools for prediction of protein structure and creation of metaprediktor, which allows the user to select the appropriate tool, according to given parameters. Testing tool is based on a dataset of proteins, which is based on the SCOP database and it is trying to be as balanced as possible to include proteins from different families and thus could best evaluate individual tools. The results of this thesis are requirements of metaprediktor and also which data and settings can be allowed and processed and how it will be implemented.
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Perovskite materials
Gavranović, Stevan ; Zmeškal, Oldřich (referee) ; Pospíšil, Jan (advisor)
This bachelor´s thesis is focused on a study of electrical properties of perovskite single crystals prepared by inverse temperature crystallization (ITC). Measurements were done on the organic-inorganic halide perovskite monocrystal MAPbBr3 and on the completely inorganic halide perovskite monocrystal CsPbBr3. Crystalline structure and chemical composition of prepared single crystals were determined using x-ray diffraction analysis. Current-voltage characteristics of perovskite monocrystals were measured using solar simulator. Hole carrier mobility were calculated from determined current-voltage characteristics using SCLC method. Furthermore, the dependencies of dielectric permittivity on frequency of alternating current were measured. MAPbBr3 single crystal showed better electrical properties (higher hole carrier mobilities) than CsPbBr3.
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Isolation and determination of the structure of hexamerin of Tribolium castaneum and TmpH protein of phi812 phage.
Valentová, Lucie ; Řezáčová,, Pavlína (referee) ; Plevka,, Pavel (advisor)
Tato práce se zabývá strukturní studií dvou proteinů: proteinu Tail morphogenetic protein H (TmpH) bakteriofága 812, který napadá Zlatého stafylokoka (Staphylococcus aureus) a hexamerinu z potemníka (Tribolium castaneum). S. aureus je jedním z nejvíce rezistentních patogenů způsobující onemocnění s vysokou morbiditou a mortalitou. Bakteriofág 812 je schopen infikovat a lyzovat 95 % kmenů S. aureus a má potenciální využití ve fágové terapii. Protein TmpH je součástí virionu tohoto fága. V rámci této práce bylo připraveno několik plazmidů nesoucích gen TmpH, které byly použity pro rekombinantní expresi proteinu v buňkách E. coli BL21(DE3). Protein byl vyčištěn afinitní a gelovou chromatografií. Pro čistý protein byly optimalizovány krystalizační podmínky. Hexamerin je nejhojnějším proteinem larev a kukel hmyzu s dokonalou proměnou. V průběhu metamorfózy hexamerin slouží jako zdroj aminokyselin. V rámci této práce byl hexamerin izolován z kukel potemníka T. castaneum. Pro stanovení struktury hexamerinu byly použity dvě metody: rentgenová krystalografie a kryo-elektronová mikroskopie. Byly optimalizovány podmínky pro růst krystalů a vypěstovány krystaly vhodné pro sběr difrakčních dat. Nicméně struktura hexamerinu byla rychleji vyřešena kryo-elektronovou mikroskopií s rozlišením 3.2 . Znalost struktury hexamerinu umožní pochopení jeho funkce v regulaci vývoje hmyzu s dokonalou proměnou.
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Structural and functional characterization of the E3 ligase BIRC6
GRATZL, Sascha
The aim of the thesis was to characterize the ubiquitin ligase BIRC6 in several contexts. The goal was to expand the knowledge on the selectivity of E1-E2 recognition, to increase the known substrate range of BIRC6, and to elucidate the molecular interactions fulfilled by BIRC6 during autophagy via interaction with GABARAP. In addition, it was aimed to gain a better understanding on the general ubiquitination mechanism in E2/E3 hybrid enzymes, to identify the ubiquitination sites of known apoptotic and autophagic substrates and to enlighten substrate recognition by BIRC6.
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Structure-assisted design of inhibitors targeting medicinally relevant enzymes
Djukic, Stefan ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee) ; Kolenko, Petr (referee)
Structure-assisted drug discovery is a powerful approach that utilizes detailed knowledge on 3D structure to design and optimize new inhibitors targeting medically relevant enzymes. X-ray crystallography is a widely used structural biology technique since it provides detailed snapshot of protein-inhibitor complex, which is used to analyze protein- inhibitor interactions. PNP plays an important role in salvage pathway of purine metabolism, it is a target in treatment of T-cell malignancies and/or parasitic infections. Our effort focused on human and M. tuberculosis PNP, and our aim was to develop new inhibitors with high selectivity and specificity. Our inhibitors are acyclic nucleoside phosphates with 9- deazahypoxanthine nucleobase that contain three moieties binding to all three regions of the active site: purine, phenyl and phosphonate moieties. The best inhibitors have IC50 values as low as 19 nM (human) and 4 nM (M. tuberculosis). The presence of short substituents at central phenyl moiety, such as methoxy and bromide group, decreases inhibitor's affinity towards human PNP, but does not affect affinity towards mycobacterial PNP. At the same time, bulky substituents, such as fluorinated phenyl ring, decrease inhibitor's affinity towards human PNP but increase affinity towards mycobacterial...
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Mechanistic and structural studies of the cGAS-STING signalling pathway
Vavřina, Zdeněk ; Maloy Řezáčová, Pavlína (advisor) ; Hudeček, Jiří (referee) ; Kolenko, Petr (referee)
The cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) signalling pathway plays a crucial role in the innate immune system. It is activated by pathogen double-stranded DNA (dsDNA) or cyclic dinucleotides, which are secondary messengers of bacteria. This activation leads to the expression of type I interferons and proinflammatory cytokines. The present dissertation examines the interaction between cGAS and its substrates and the relation between the STING protein and its agonists from a mechanistic and structural point of view. The enzyme cGAS is a metazoan intracellular sensor of dsDNA. Upon its binding to DNA, it synthesizes the cyclic dinucleotide 2′,3′-cGAMP, which activates the adaptor protein STING. Besides 2′,3′-cGAMP, STING can also be activated by 3′,3′-cyclic dinucleotides that serve as secondary messengers in bacteria. We investigated various dinucleotide cyclases to better understand their substrate specificity and utilized them for the preparation of novel cyclic dinucleotides activating STING. As the most appropriate for the preparation of 2′,3′-cyclic dinucleotides, we identified mouse cGAS. Additionally, we utilized the enzymes DncV from Vibrio cholerae and DisA from Bacillus thuringiensis for the synthesis of 3′,3′-cyclic dinucleotides. These enzymes exhibit...
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Structure and Function of the C-terminal Domain of the HsdR Subunit from the Type I Restriction-Modification System EcoR124
GRINKEVICH, Pavel
The Type I restriction-modification enzyme EcoR124 is a pentameric complex consisting of one specificity subunit, two methylation subunits and two motor subunits (HsdR) that can recognize specific DNA sequences and perform double-stranded DNA cleavage and modification. The HsdR subunit is responsible for ATP-dependent DNA translocation and DNA cleavage. Even though the first crystal structure of HsdR was obtained ten years ago, a large part of the C-terminus has not been resolved in any HsdR structures to date. This dissertation aims to elucidate its role within the HsdR subunit and the whole pentameric complex by solving the structure of the C-terminus by means of X-ray diffraction crystallography and explore its function using biochemical, microbiological, bioinformatical and computational methods.
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Preparation and study of human lymphocyte receptor LLT1
Bláha, Jan ; Vaněk, Ondřej (advisor) ; Konvalinka, Jan (referee)
Natural killer (NK) cells are an intensively studied part of immune system, possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. Recent research in their C-type lectin-like receptors repertoire has shown that ligands of some of these previously orphan receptors lie within their own family, describing a lectin-lectin interaction. This is the case of human inhibitory receptor NKRP1 (gene KLRB1) and its ligand LLT1 (gene CLEC2D). Previous studies have shown that overproduction of LLT1 in cancer cells or lower production of NKRP1 in NK cells is connected to cancerous manifestations. This master's thesis shows a successful production of the extracellular part of LLT1 utilizing a mammalian expression system based on transient transfection of modified human embryonic kidney (HEK) cell lines. It was found that the five cystein residues contained within the lectin domain of LLT1 tend to cause misfolding and formation of aggregates. Stabilization of the domain was achieved by restoration of the sixth cystein residue at the evolutionary conserved...
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Structure-assisted development of a continuous carboxypeptidase assay
Rakhimbekova, Anastasia ; Bařinka, Cyril (advisor) ; Bouřa, Evžen (referee)
Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...
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