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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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Utilization of chemical cross-linking for studying intermediate filaments organization
Dlabolová, Lada ; Novák, Petr (advisor) ; Sabó, Ján (referee)
Intermediate filaments are cytoskeleton components formed by a large family of fibrous proteins specifically expressed in nearly all differentiated cells. Under physiological conditions, they spontaneously assemble into fibers in a process that involves several stages in the organization of subunits. These fibers provide elastic properties to the cells, allowing them to maintain their structural and mechanical integrity. While the structure of other cytoskeletal components is now well researched, detailed information on the structure of intermediate filaments at various stages of assembly is still not available. Thus, new insights into the structure of these proteins could be of great benefit in understanding of various pathological mechanisms associated with changes in their expression in cells. This thesis studies interactions of dimeric subunits in the tetrameric assembly of vimentin, class III protein of intermediate filaments. By chemical cross-linking of isotopically labeled and unlabeled tetrameric vimentin mixture, followed by proteolytic cleavage and mass spectrometry analysis, interdimeric, intradimeric and intrapeptide cross-linking products were identified. Quantification yielded information on interdimeric and intradimeric distance constraints, which allow the characterization of a...
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Study of the interaction between DNA and transcription factors using mass spectrometry.
Slavata, Lukáš ; Man, Petr (advisor) ; Obšil, Tomáš (referee)
Transcription factors play crucial regulatory role within the cell and the entire multicellular organism. The important factor is its ability to interact with other regulatory proteins and DNA. Despite the fact that a large part of the interaction network is already documented, detailed information on the structure and dynamics of protein-protein and protein-DNA complexes is still scarce. In this thesis we focused on the possibility of studying conformational changes given by the transcription factor-DNA complex formation using the methods of structural mass spectrometry: hydrogen/deuterium exchange and chemical crosslinking. As a model, we chose a transcription factor FOXO4 which DNA binding domain is structurally well characterized both in free form and in the complex with DNA.
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