Utjecaj fosfata na promjenu konformacije aktivnih mjesta enzima purinske nukleozidne fosforilaze iz bakterije Helicobacter pylori

master's thesis

• Title (english): The influence of phosphate on conformation change of the active sites of purine nucleoside phosphorylase from Helicobacter pylori
• Author: Marta Bošnjaković
• Mentor: Zoran Štefanić (mentor)
• Mentor: Biljana Balen (sumentor)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Vesna Benković (član povjerenstva)
• Committee member: Silvija Černi (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-03-12, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Hp-PNP (purinska nukleozidna fosforilaza iz Helicobacter pylori) je ključni enzim pomoćnog metaboličkog puta purina u bakteriji H. pylori. Katalizira fosforolitičko cijepanje glikozidne veze u purinskim nukleotidima. Protein je homoheksamer sa 6 aktivnih mjesta u dvije moguće konformacije: zatvorenoj (neophodnoj za odvijanje katalize) i otvorenoj. Cilj rada je bio provjeriti dolazi li do vezanja fosfata (jednog od dva supstrata Hp-PNP) u aktivna mjesta enzima, namakanjem kristala u otopinama s... More rastućom koncentracijom fosfata i utjecaj tog vezanja na konformaciju aktivnog mjesta. Opisan je postupak transformacije bakterija Escherichia coli vektorom s ugrađenim deoD genom za Hp-PNP protein, prekomjerna ekspresija, pročišćavanje kromatografskim metodama, mikrokristalizacijski eksperimenti, te određivanje i utočnjavanje strukture proteina metodom rendgenske difrakcije. Određene su dvije strukture dobivene iz različitih koncentracija fosfata. U strukturi s višom koncentracijom fosfata došlo je do vezanja fosfata u 5 od 6 aktivnih mjesta, ali niti u jednom nije došlo do zatvaranja aktivnog mjesta. U strukturi s nižom koncentracijom fosfata nije došlo do njegovog vezanja i također su sva aktivna mjesta ostala otvorena. Ove strukture su prve određene strukture Hp-PNP u kojima vezanje fosfata nije dovelo do promjene konformacije, čime je pokazano da to nije dovoljan uvjet za zatvaranje aktivnog mjesta kod Hp-PNP enzima. Less

Abstract (english)

Hp-PNP (purine nucleoside phosphorylase from Helicobacter pylori) is the key enzyme of salvage pathway of purines in H. pylori. It catalyzes phosphorolitic cleavage of the glycosidic bond in purine nucleotides. The protein is a homohexamer with 6 active sites in two possible conformations: closed (essential for catalysis) and open. The aim of the study was to determine phosphate binding (one of two substrates of Hp-PNP) by soaking the crystals in solutions with increasing phosphate... More concentration, and its effect on the conformation of the active sites. This thesis describes transformation of Escherichia coli bacteria with deoD gene coding for Hp-PNP, protein over-expression, cromatography purification, micro-crystallization experiments and X-ray structure determination and refinement. Two structures were obtained from different phosphate concentrations. In the structure with higher phosphate concentration, phosphate binding occurred in 5 out of 6 active sites, but none of the active sites were closed. In the structure with lower phosphate concentration there was no binding and all active sites remained open. These structures are the first determined Hp-PNP structures in which the binding of phosphate did not lead to a change of conformation, thus indicating that this is not sufficient condition for closing of the active site in Hp-PNP enzyme. Less

• Keywords: humani patogen; pročišćavanje proteina; kristalizacija proteina; rentgentska strukturna analiza; heksamerni protein
• Keywords (english): human pathogen; protein puification; protein crystalisation; X-ray diffraction; hexamer protein complex
• Language: croatian
• URN:NBN: urn:nbn:hr:217:772159
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2018-06-12
• Created on: 2018-05-04 11:28:28