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Modulation of interactions between interleukins and their receptors
Nepokojová, Tereza ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee)
Scaffolds are proteins with high conformational stability, allowing us to implement multiple mutations into specific parts of the protein. Even with these mutations, the structural integrity of the protein is maintained as well as its physical-chemical properties. These mutations give the specific scaffold new properties. In most cases it is the binding specificity towards previously chosen target. The biggest advantages of scaffolds are their small size, stability, low-cost manufacturing, and easiness of preparation. Scaffold utilized in this thesis is unique for having two binging surfaces designed on which it can be mutated. Each of those two surfaces can be separately mutated to develop a binging site for two different proteins. In our case these mutations led to binding two nonidentical receptors of a human cytokine. Mutations are made with a use of yeast display, one of the methods of directed evolution. The main focus of this thesis is changing an expression system of the binding proteins from the yeast system to a bacterial one, their production and purification followed by characterization of those binding proteins using biophysical methods. These methods were used to evaluate structural and thermal stability, and binding affinity to both receptors of the beforementioned binding proteins....
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Biophysical characterization of protein libraries composed of different amino acid repertoires
Neuwirthová, Tereza ; Hlouchová, Klára (advisor) ; Ptáček, Jakub (referee)
This study is part of a project which aims to understand evolution of genetic code together with structural and functional analysis of prebiotic proteins. The repertoire of amino acids in the first proteins was probably developing in time and it influenced the development of structure and function of today's proteins. First amino acid alphabet was apparently only half of the size of present alphabet, which contains twenty amino acids. These ten amino acids were probably prebiotically available from endogenous and exogenous sources. This work includes cell-free expression and purification of two randomized protein libraries (containing approximately 1011 variants) with various amino acid composition and following comparison of their propensity to form secondary (using circular dichroism) and tertiary (using proteolytical analysis of sequences) structures. First library contains only ten probably prebiotically available amino acids; second library contains all twenty amino acids in today's genetic code. This project could help us understand benefits of genetic code expansion in terms of developing structure in protein sequences. The whole research could theoretically contribute a few basic questions not only in the fields of protein evolution but also in areas of synthetic biology or protein...
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Structural characterization of selected random protein sequences with high disorder content
Ptáčková, Barbora ; Hlouchová, Klára (advisor) ; Šilhán, Jan (referee)
An infinitesimal fraction of the practically infinite sequence space has achieved enormous functional diversity of proteins during evolution. Intrinsically disordered proteins (IDPs) which lack a fully defined three-dimensional structure are the most likely precursors to today's proteins because of their flexible conformation and functional diversity. But how have these proteins evolved into often rigid and highly specialized protein structures? This evolutionary trajectory has the greatest support in the theory of induced fold whereby the development of the structure was mediated by the interaction and coevolution of primordial unstructured proteins with different cofactors or RNA molecules. Although some random sequences from the sequence space which is not used by nature are also able to form folded proteins the more suitable candidates for evolution of structure and function appear to be random sequences with a high content of disordered which have low aggregation propensity. The selected random protein sequences with high disorder content have been structurally characterized in this work for their further use in evolutionary studies. Three artificial proteins were selected from a random-sequence library based on previous study in our laboratory. In the present work they were purified and...
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Molecular evolution of meiosis in diploids and tetraploids of Arabidopsis arenosa
Holcová, Magdalena ; Schmickl, Roswitha Elisabeth (advisor) ; Mozgová, Iva (referee)
Meiosis is functionally conserved across eukaryotes, thus not expected to vary considerably among different species, and even less so among lineages within a species. However, recent studies showed that this is not necessarily the case in Arabidopsis arenosa. Genome scanning identified an excess differentiation in meiosis genes between A. arenosa diploids and tetraploids, interpreted as meiosis adaptation to the whole genome duplication in tetraploids and differentiation was also found between two diploid lineages. Thus, I present a population-based analysis of positive selection acting on meiosis proteins across multiple lineages of A. arenosa. I showed that meiosis proteins were under positive selection in all diploid lineages, mainly in the Pannonian and South-eastern Carpathian lineage. The evidence for positive selection in diploid lineages suggested differential pathways of meiosis adaptations in the species, probably reflecting the necessity to adapt to local environments, among all to temperature. The highest enrichment of amino acid substitutions (AASs) under positive selection was identified in tetraploids, in consistence with previous genome-scan results. As several interacting meiosis proteins were under positive selection in the same A. arenosa lineage, I hypothesize that the close...
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Expression and characterization of selected never-born proteins
Šimonová, Johana ; Hlouchová, Klára (advisor) ; Pavlíček, Jiří (referee)
This bachelor thesis deals with the study of random polypeptide sequences and occurence of secondary structure in random sequence space. Sequences for experimental characterization were selected from library containing 104 random sequences on the basis of (i) either predictions of secondary structure and disorder occurence and prediciton of solubility or (ii) a completely random selection. Eight proteins studied in this thesis belong to the second option - i.e. they were selected from the in silico generated library completely randomly. First, expression and solubility was analysed for these eight proteins. In addtion, one of the proteins was selected for further analysis. The thesis involves optimization of its expression and purification as well as its detailed biophysical characterization. (In Czech) Key words: protein evolution, protein structure, random sequences
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