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Bioinformatic analysis of protein/DNA interactions
Božíková, Paulína ; Schneider, Bohdan (advisor) ; Hašek, Jindřich (referee)
In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.
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Structural studies of 14-3-3 protein complexes and their stabilization by small molecule compounds
Lentini Santo, Domenico ; Obšil, Tomáš (advisor) ; Brynda, Jiří (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions (PPIs) play a crucial role in almost all biological processes. Many proteins require a number of dynamic interactions with other proteins and/or biomolecules to function. Proteomic studies have suggested that human protein-protein interactome consists of several hundred thousands of protein complexes. A detailed insight into these PPIs is essential for a complete understanding of the processes mediated by these protein complexes. Because many PPIs are involved in disease-related signaling pathways, such PPIs are important targets for pharmaceutical interventions, especially in situations where a more conventional target (e.g. the active site of an enzyme, the binding site of a receptor) cannot be used. This doctoral thesis focuses on 14-3-3 proteins, a family of eukaryotic adaptor and scaffolding proteins involved in the regulation of many signaling pathways. The 14-3-3 proteins function as interaction hubs and critical regulators of many enzymes, receptors and structural proteins. The main aim was to structurally characterize selected 14-3-3 protein complexes and investigate their stabilization by small molecule compounds. Using combination of protein crystallography, differential scanning fluorimetry, fluorescence polarization and analytical ultracentrifugation, the...
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Structural and functional study of viral RNA polymerases
Dubánková, Anna
Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...
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Study of structural features of single stranded DNA by biophysical techniques and crystallography
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Pavlíček, Jiří (referee)
DNA is the fundamental molecule in all domains of life, its role in heredity is well established. Although the famous double helical complementary form is indispensable for replication mechanism DNA can occupy wide range of conformations. In the past studies performed in the laboratory, DNA oligomers related to single stranded bacterial Repetitive Extragenic Palindromic (REP) showed spectral behavior suggesting complex equilibria including double helical, hairpin, and tetraplex conformations. The studies presented in this thesis extended the scope of analyzed sequences and employed circular dichroism spectroscopy and X-ray crystallography. We report spectral data and X-ray structures of three successfully crystalized oligonucleotides. All three structures acquire double helical architecture with two consecutive T- T mismatches in the center. To improve the convergence of the refinement process of the crystal structures we used novel dinucleotide conformational classes, NtC classes. The NtC class classification was also used to analyze geometries of selected non-canonical base pairs in all DNA crystal structures in the Protein Data Bank. We measured the fit between geometries of the dinucleotides involved in the non-canonical base pairing and the NtC classes and correlated this fit to the electron...
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Stability of dispersoids in aluminium alloys at elevated temperatures.
Králík, Rostislav ; Cieslar, Miroslav (advisor) ; Michalcová, Alena (referee)
Hliníková slitina AA8079 připravena plynulým odléváním mezi válce je běžně používána pro výrobu tenké potravinářské fólie. Vzhledem k použité metodě odlévání a složení slitiny je struktura litého materiálu značně nehomogenní a obsahuje intermetalické fáze, které se shlukují v eutektických koloniích. Litý materiál tak vyžaduje tepelné zpracování před dalšími kroky výroby. Mikrostruktura materiálu po homogenizačních žíháních na různých teplotách je rozdílná, což ovlivňuje další zpracování. Po homogenizaci je materiál válcován což způsobuje snížení tažnosti. Rekrystalizační žíhání je vyžadováno po zválcování na střední tloušťku před finálním válcování, aby byla tažnost obnovena. Chování materiálu během rekrystalizačního žíhání je ovlivněno přítomnými fázemi, jejich velikostí a rozdělením. Byl studován vliv mikrostruktury po homogenizaci na rozdílných teplotách na rekrystalizaci, byla vyhodnocena kinetika rekrystalizace a byly identifikovány dva hlavní mechanismy ovlivňující rekrystalizaci - částicemi stimulovaná nukleace a Zenerův tlak.
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Metastable iron thin films for magnetic metamaterials
Holobrádek, Jakub ; Man, Ondřej (referee) ; Urbánek, Michal (advisor)
Magnetické nanostruktury mají zajímavé vlastnosti, které umožňují jejich aplikace v základním výzkumu i průmyslu. Jednou z těchto vědeckých disciplín je i magnonika - výzkumný obor, který se zabývá fyzikou spinových vln, které lze použít v nediskrétních výpočtech s nízkými ztrátami energie. Výroba magnetických struktur fokusovaným iontovým svazkem (FIB) je alternativní metoda k běžně používaným litografickým metodám. Materiál použitý v této práci - metastabilní železo - je schopen při ozáření iontovým svazkem podstoupit fázovou transformaci z paramagnetické plošně centrované kubické krystalové struktury na feromagnetickou fázi s prostorově centrovanou kubickou krystalovou strukturou. Jednou z vlastností, která ovlivňuje šíření spinových vlny, je magnetická anizotropie. Tato práce představuje vliv depozičních podmínek v ultra vysokém vakuu během přípravy metastabilní železné vrstvy na magnetickou anizotropii struktur vytvořených pomocí FIB do tohoto filmu. Dále prezentujeme souvislosti mezi parametry FIB, krystalografickými vlastnostmi výsledných struktur a jejich magnetickou anizotropií.
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Structural and functional study of viral RNA polymerases
Dubánková, Anna
Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...
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Structural and functional study of viral RNA polymerases
Dubánková, Anna ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Plevka, Pavel (referee)
Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...
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Study of single stranded DNA by biophysical techniques
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Novák, Petr (referee)
DNA is the fundamental functional molecule of all domains of life. One of its characteristics is the ability to self-associate to form a double stranded helix. Nevertheless, even single stranded DNA can form non-canonical structures such as hairpins, triplexes or tetraplexes. One of the interesting single stranded sequences are REP elements. These sequences form a part of bacterial transposable elements, so-called insertion sequences, which can be found in a great number of copies in wide range of bacterial species. This thesis studies structure and properties of selected REP sequences. It presents the results of spectral measurements by technique of circular dichroism, melting curves from differential scanning calorimetry, and monocrystal diffraction data. Key words Single-stranded DNA, crystallography, circular dichroism, calorimetry, transpozone, REP elements, insertion sequences.
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Krystalizační studie nově izolované halogenalkandehalogenasy DgaA z \kur{Glacieocola agarilytica} NO2
BERKOVÁ, Ivana
This bachelors thesis is focused on crystalization study of newly prepared halogenalkanedehalogenase DgaA from bacteria Glaciecola agarylitica NO2. Main target of this work is getting acquianted with methods of protein crystalization and usage of those methods for preparation of suitable DgaA protein crystals of that will be used for X-ray structural analysis. Results from difraction analysis of DgaA crystals will be starting point for further research focused on structure determination and and description of protein function.
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