Badania krystalograficzne 1,3-β-glukanazy z Solanum tuberosum
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Date
2013-05-24
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Crystallographic studies of 1,3-β-glucanase from Solanum tuberosum
Abstract
W ramach pracy doktorskiej przedstawiłam badania krystalograficzne 1,3-β-glukanazy z Solanum tuberosum
(ziemniak, odmiana Désirée). Enzym ten należy do rodziny 17 hydrolaz glikozydowych (GH17) i posiada typ
pofałdowania cylindra (β/α)8, znany również pod nazwą „TIM barrel”. Oznaczone przeze mnie dwie struktury
krystaliczne białka natywnego pozwoliły na wyznaczenie najbardziej zmiennych i zarazem mobilnych fragmentów
strukturalnych w roślinnych 1,3-β-glukanazach. Analiza upakowania cząsteczek w kryształach potwierdziła,
że obecność znacznika histydynowego nie zaburzyła struktury przestrzennej enzymu oraz pomogła w otrzymaniu
wysokiej jakości kryształów. Poddanie zmutowanej formy białka kokrystalizacji z naturalnym substratem, pozwoliło
na zaobserwowanie po raz pierwszy dla enzymów z rodziny GH17 oddziaływań pomiędzy sacharydami a białkiem.
The thesis describes crystallographic studies of 1,3-β-glucanase from Solanum tuberosum (potato, cultivar Désirée). The enzyme belongs to family 17 of glycoside hydrolases (GH17) and has the TIM-barrel (β/α)8 folding pattern. The discussion presented in the thesis is based on four high-resolution crystal structure determinations, two for the (recombinant) protein in its native sequence, and two for its active-site mutant in complex with carbohydrate products of the hydrolysis reaction. The two crystal structures of the native protein allowed me to identify the most variable and most mobile regions of the polypeptide chain of plant 1,3-β-glucanases. The analysis of crystal-packing contacts confirmed that the presence of an intact histidine tag, used as a chromatographic purification artifact, did not changed the overall fold of the protein, but instead - promoted the formation of very high quality crystals. In the second part of the thesis, cocrystallization of the mutated enzyme with a natural substrate has resulted in the first crystallographic characterization of a protein/saccharide complex for a GH17 enzyme.
The thesis describes crystallographic studies of 1,3-β-glucanase from Solanum tuberosum (potato, cultivar Désirée). The enzyme belongs to family 17 of glycoside hydrolases (GH17) and has the TIM-barrel (β/α)8 folding pattern. The discussion presented in the thesis is based on four high-resolution crystal structure determinations, two for the (recombinant) protein in its native sequence, and two for its active-site mutant in complex with carbohydrate products of the hydrolysis reaction. The two crystal structures of the native protein allowed me to identify the most variable and most mobile regions of the polypeptide chain of plant 1,3-β-glucanases. The analysis of crystal-packing contacts confirmed that the presence of an intact histidine tag, used as a chromatographic purification artifact, did not changed the overall fold of the protein, but instead - promoted the formation of very high quality crystals. In the second part of the thesis, cocrystallization of the mutated enzyme with a natural substrate has resulted in the first crystallographic characterization of a protein/saccharide complex for a GH17 enzyme.
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Wydział Chemii:Zakład Krystalografii
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Keywords
Hydrolaza glikozydowa, Glucoside hydrolase, Roślinne białko odpowiedzi na stres klasy PR-2, Plant pathogenesis-related protein of class 2, Oddziaływania białko-węglowodany, Protein-carbohydrate interactions, Kompleks białko-ligand, Protein-ligand complex