|
|
Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
|
|
|
Development of alternative proteolysis for protein identification
Jirečková, Barbora ; Man, Petr (advisor) ; Hnízda, Aleš (referee)
5 Abstract Methods of protein research based on mass spectrometry have become increasingly significant in the last few years. The proteolytic step is essential in a bottom-up approach, determining the success and relevance of identification and characterization of a protein and also the sequence coverage and spatial resolution of HDX-MS protocols. Conventionally used proteases however have their limitations and therefore efforts are being made to find alternatives that have, above all, different cleaving preferences and activity profiles. AnPEP protease (Aspergillus niger prolyl endoprotease) has the advantages of both characteristic cleavage in the presence of proline (unlike many other proteases) and easy accessibility due to its commercial obtainability in large quantities. This thesis examines the optimization of cleavage using AnPEP protease obtained from commercially prepared Gluten Rid with Tolerase G. Firstly AnPEP cleavage was tested on model protein (bovine carbonic anhydrase 2) under various conditions (temperature, pH, concentration of protease on a carrier or in solution). The objective was to find out how various conditions influence cleavage efficiency and preferences of this protease. It was discovered that AnPEP protease is suitable for hydrogen/deuterium exchange coupled to mass...
|
|
|
Study of conformations and conformational changes of proteins using mass spectrometric methods.
Kádek, Alan ; Man, Petr (advisor) ; Šebela, Marek (referee) ; Hnízda, Aleš (referee)
Mass spectrometry (MS) techniques have, over the last twenty years, found their stable place in the structural biology toolkit. They are not only employed to provide information on the protein primary sequence, but are increasingly used to probe higher orders of protein structure as well. They may not boast the atomic resolution and the ability to directly provide structural coordinates, but on the other hand suffer from very few experimental limitations as they are able to work under native conditions in solution, provide data fast, with low sample consumption and for proteins and complexes of vastly differing sizes. Perhaps most importantly, they may often be employed to study conformational dynamics of proteins and can thus complement other methods with higher spatial resolution in integrative structural biology approaches. The main focus of this Ph.D. thesis was hydrogen / deuterium exchange coupled to MS (HXMS), which is one of the most widespread structural MS methods. Recombinantly produced aspartic protease nepenthesin-1 from Nepenthes pitcher plants was characterized, immobilized and extensively tested with the intention to expand the portfolio of aspartic proteases in HXMS workflow and to improve the spatial resolution of the technique. Following successful implementation of nepenthesin-1...
|
|
|
Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
|
guest ::
