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Development of pulse labelling technology for studying the dynamics of protein complexes.
Fiala, Jan ; Novák, Petr (advisor) ; Preisler, Jan (referee) ; Sosic, Alice (referee)
(IN ENGLISH) Structural mass spectrometry (MS) is an evolving field of structural biology introducing novel techniques for the characterization of biomolecules. Although MS-based techniques only can provide "low-resolution" information compared to standard high-resolution techniques representing by X-ray crystallography, cryo-electron microscopy or nuclear magnetic resonance, its uniqueness lies in the ability to easily obtain structural information about various biomolecules in their native or native-like environment. By employing various approaches, from protein covalent labelling through chemical cross- linking to ion mobility, structural MS provides insight into the structure and dynamics of proteins and their complexes over a broad timescale. This thesis is dedicated to the development of novel structural MS approaches based on pulse covalent labelling and chemical cross-linking. Employing the developed quench-flow microfluidics apparatus, we performed footprinting experiments on proteins and protein complexes in timescale from a few microseconds to single seconds. Specifically, fast photochemical oxidation of proteins (FPOP) and novel fast fluoro alkylation of proteins (FFAP) techniques were utilized to track structural changes of myoglobin upon release of the prosthetic heme group....
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Structural NMR studies of protein complexes
Hexnerová, Rozálie ; Veverka, Václav (advisor) ; Hrabal, Richard (referee) ; Hlouchová, Klára (referee)
Protein-protein interactions are involved in various biological processes and detailed characterization of their structural basis by the means of structural biology is often instrumental for rigorous understanding of underlying molecular mechanisms. This information is important not only for fundamental biology but also plays an important role in search for sites amenable for therapeutic intervention. Nuclear magnetic resonance spectroscopy is alongside X-ray crystallography and single-particle cryo-electron microscopy one of the key high-resolution techniques in structural biology. Although its applicability to larger systems has a well-known physical limit, it offers unique capabilities in addressing highly dynamic or inherently heterogeneous systems. In this doctoral thesis, the solution-based NMR approach was used for detailed structural characterization of selected biologically important proteins and their complexes that provided important insights into their biological roles. In three distinct projects, I (i) studied the relationship between the structural effects of particular modifications in the insulin-like growth factor II (IGF-II) and their selectivity to the insulin axis receptors; (ii) the specific binding mechanism of the SH3 domain from the Crk-associated substrate (CAS); (iii) and...
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Influence of peptides/proteins produced by Microcystis aeruginosa on coagulation process
Barešová, Magdalena ; Pivokonský, Martin (advisor) ; Janda, Václav (referee)
The aim of the diploma thesis is to analyze the mechanisms involved in the coagulation of peptides and proteins contained in cellular organic matter produced by Microcystis aeruginosa, and to describe their influence on the coagulation of hydrophobic kaolin suspension. According to the results of jar tests, the coagulation effectiveness and removability of COM peptides/proteins and kaolin particles are heavily dependent on pH value which determines charge characteristics of peptides/proteins, kaolin and hydrolysis products of coagulant and therefore the prevailing mechanisms of interactions between them. Efficient coagulation and the highest removal of COM peptides and proteins were achieved in the pH range of 4-6 due to charge neutralization of peptide/protein negative surface by positively charged hydrolysis products of ferric coagulant. Peptides and proteins contributed to the coagulation of kaolin particles under the reaction conditions mentioned above, too. Charge neutralization and adsorption were found to be the dominant coagulation mechanisms under these conditions. At a low COM/Fe concentration ratio (COM/Fe < 0.33), adsorption of peptides/proteins onto ferric oxide-hydroxide particles, described as the electrostatic patch model, enabled the coagulation in the pH range of 6-8. On the...
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