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Utilization of pulse labelling techniques for studying the dynamics of proteins and protein complexes
Polák, Marek ; Novák, Petr (advisor) ; Obšil, Tomáš (referee) ; Vrbacký, Marek (referee)
(In English) Mass spectrometry (MS) techniques are routinely used to probe the structure and dynamics of proteins and protein complexes. Although MS techniques lack the high resolution of data provided by X-ray crystallography, NMR, or cryo-EM, they excel in providing insights into analyte dynamics, structure, and interactions with other components, such as ligands. This doctoral thesis presents a contribution to the field of structural biology employing and extending covalent labelling approaches, namely Fast Photochemical oxidation of Proteins (FPOP) and oxidation by singlet oxygen (1 O2). These approaches were followed to study the structure, dynamics, and interaction of proteins, nucleic acids, and protein-DNA complexes in solution. Initially, FPOP was used to investigate the interaction interface of FOXO4 and DAF16-DNA response element and to show the possibilities of analyzing such a complex using both 'bottom- up' and 'top-down' approaches. Furthermore, an isotope depletion strategy combined with multiCASI-ECD proved effective in delivering structural information with the highest possible resolution for mapping protein-DNA interfaces. This research showcases how information derived from structural proteomic methods can guide the construction of in-silico models for protein-DNA complexes with...
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Mass spectrometry in structural proteomics: study of viral protein complexes
RAVI, Anirudhan
This thesis investigated the avian reovirus sigma NS protein and its interaction with RNA using mass spectrometric methods. The sigma NS structure is still not known; thus, hydrogen-deuterium exchange and cross-linking coupled to mass spectrometry were employed to understand protein conformation. This research provides insights into protein-RNA interactions and the obtained data will be used in future work to predict protein structure.
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Structural characterization of a model heme-containing oxygen sensor
Tajovská, Eva ; Martínková, Markéta (advisor) ; Ryšlavá, Helena (referee)
One subgroup of hemoproteins are heme-based gas sensors, which are able to detect biatomic gas molecules in their immediate surroundings. Upon binding of a gas molecule to the heme iron in a sensor domain of these proteins or, conversely, upon its dissociation from the heme iron, the signal is then transmitted from the sensor domain to a functional domain and subsequent regulation of important cellular functions occurs. Understanding the regulatory mechanism of gas sensors is key to potentially manipulating their function. Such knowledge would then allow the use of heme-based gas sensors as therapeutic targets for the development of next-generation antibiotics, if we take into account their presence in pathological bacteria. The diploma thesis focuses on a model heme-based gas sensor, the oxygen sensor EcDOS from E. coli, and its apoform, EcDOS His77Ala. Both proteins were prepared by recombinant expression and purification, and subsequently spectrophotometrically characterized. Using gel permeation chromatography, the oligomeric states of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS His77Ala were determined under different conditions (different temperatures of protein incubation, presence of c-di-GMP substrate etc.). Furthermore, the structural dynamics of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS...
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Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
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Structural characterization of interaction between transcription factors and DNA
Filandrová, Růžena ; Novák, Petr (advisor) ; Vondrášek, Jiří (referee) ; Wimmerová, Michaela (referee)
Structural characterization of interaction between transcription factors and DNA Mgr. Růžena Filandrová Abstract Transcription factors are proteins that mediate gene expression regulation through interactions with DNA and other factors. They allow a cell to respond to various stimuli and play a crucial role in many biological processes such as control of cell cycle progression, differentiation of cells during development or immune response. To understand these processes, the knowledge of the transcription factors 3D structure together with the mechanism of their interaction with DNA is essential. However, some of the typical features of transcription factors, such as is for example the presence of intrinsically unstructured regions, make the 3D structure determination by the commonly used high resolution methods challenging. Therefore, utilization of complementary methods like structural mass spectrometry (MS), which was used in this thesis, might prove to be beneficial to explore the structural basis of the transcription factor-DNA interaction. In first part of this work, a set of structural mass spectrometry methods with the main focus on hydrogen/deuterium exchange mass spectrometry (HDX-MS) was optimized and tested on two transcription factor-DNA complexes and their DNA binding motifs and proved to be...
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Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
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