This study is part of a project which aim is solving the structure of the catalytic domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is the preparation of new constructs of yeast Nth1 and optimization of their purification protocols, the selection of the ideal buffer for crystallization trials using the method of differential scanning fluorimetry (DSF) and at last the protein crystallization. Another part of the thesis is the measurement of the enzymatic activity of pNth1 WT in the presence of Bmh1 protein, verification of trehalose binding to the selected constructs of Nth1 using differential scanning fluorimetry (DSF), thermoforesis (MST) and further crystallization with trehalose. Neutral trehalase is highly conserved trehalase that has been found in a wide variety of organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and hydrolytically cleave trehalose into two glucose molecules. Trehalose is a naturally occurring non-reducing disaccharide serving in yeast cells a source of carbon and energy as well as protection against stress conditions such as a thermal shock. Trehalose hydrolysis is essential for flying insects, because it is present as the main sugar component of insect haemolymph, therefore trehalase inhibitors...
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:342003 |
| Date | January 2016 |
| Creators | Šmídová, Aneta |
| Contributors | Obšilová, Veronika, Jiráček, Jiří |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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