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Preparation and characterization of neutral trehalase for structural studies
Šmídová, Aneta ; Obšilová, Veronika (advisor) ; Jiráček, Jiří (referee)
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...
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Stability of the catalytic domain of neutral trehalase studied by differential scanning fluorimetry
Šmídová, Aneta ; Obšilová, Veronika (advisor) ; Kacířová, Miroslava (referee)
This bachelor thesis is part of a project aiming for the crystallization of trehalase domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is to optimize the purification protocol of two different constructs of yeast Nth1 and determine the optimal buffer for crystallography of these constructs using the differential scanning fluorimetry DSF. Trehalases are important, highly conserved enzymes found in many organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and their common feature is hydrolytic cleavage of trehalose molecule into two glucose subunits. Trehalose is a naturally occurring non-reducing disaccharide which serve in yeast cells as storage carbohydrate and energy and stress metabolite. Trehalose and its hydrolysis is very important in the life cycle of the insect, as it is present as the main sugar component of insect hemolymph, so some trehalase inhibitors could be used as insecticides. By this time, however, only one structure of trehalase from a prokaryotic organism Escherichia coli was solved. For this reason it is very important to solve the structure of trehalase from a eukaryotic organism. Keywords: Nth1, expresion, purification, DSF Key subjects: Protein biochemistry, biophysical chemistry
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