Vrednotenje DNA-specifičnosti in termične stabilnosti PhD

Kranjc, Jaka (author), Ilaš, Janez (mentor), Loris, Remy (comentor)

Bakterijska persistenca je pojav, pri katerem se celicam spremenijo morfološke lastnosti in vstopijo v fazo mirovanja. S tem močno povečajo svojo odpornost proti zunanjim stresnim dejavnikom (kot so protimikrobne učinkovine, povišana ionska jakost, pomanjkanje hranil, UV sevanje), saj le-ti ne morejo vstopiti v celico. Bakterijski sistemi toksin-antitoksin so ena izmed metabolnih poti, ki v bakterijski celici sprožijo persisterski odziv. Predmet te magistrske naloge je protein Phd, del sistema toksin-antitoksin Phd /Doc, ki se nahaja na bakteriofagu P1 v bakterijah E. coli. Cilj našega dela je bil odkriti vpliv mutacij v palindromnem zaporedju operatorja na vezavne lastnosti z antitoksinom Phd. Hkrati pa smo proučevali tudi vpliv ionske jakosti na sekundarno strukturo proteina ter na njegovo termično stabilnost. Za pridobitev čistega proteina Phd smo izvedli čiščenje z uporabo afinitetne kromatografije. Pri tem smo celični lizat bakterij E. coli, v katerem sta se nahajala Phd in Doc v obliki kompleksa, nanesli na kolono ter ju ločili z denaturacijo. Naslednji korak je bil analiza sekundarne strukture proteina v pufrih različne ionske jakosti z metodo CD spektroskopije. Da bi zagotovili optimalne pogoje pri izvedbi izotermalne titracijske kalorimetrije, smo poskusili določiti pufer, v katerem je protein Phd najbolj termično stabilen. V ta namen smo izvedli test toplotne stabilnosti. Vpliv mutacij v palindromičnem zaporedju DNA, na katerega se veže Phd, smo analizirali z uporabo izotermalne titracijske kalorimetrije. Z izbrano metodo določanje toplotne stabilnosti proteina ni bilo uspešno. Pri tem nas je verjetno oviral neurejen repni del proteina, ki nase veže barvilo, ki smo ga uporabili pri testu toplotne stabilnosti. Z meritvami, pridobljenimi z izotermalno titracijsko kalorimetrijo, smo ugotovili, da vsakršne mutacije v oligonukleotidnem zaporedju negativno vplivajo na vezavo Phd. S pomočjo spletne podatkovne baze Protein data bank smo pridobljene rezultate ovrednotili v povezavi z že znano kristalno strukturo proteina Phd v kompleksu z operatorjem.

• Language: Slovenian
• Keywords: proteini, toksin-antitoksin, izotermalna titracijska kalorimetrija, termodinamika, interakcije z DNA
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-115994
• Publication date in RUL: 06.05.2020

Secondary language

• Language: English
• Title: Characterization of DNA-specificity and thermal stability of the PhD protein

Abstract

Bacterial persistence is an endogenous survival mechanism employed by bacteria to persevere in hazardous environments. In the persister state cells are not inherently resistant to the hazardous environmental effects (for example antibiotics), but due to their phenotypical attributes their tolerance against the stress factors is heightened. Understanding the pathways that govern the processes of bacterial persistence could lead to novel antibiotic targets. Toxin-antitoxin systems are mechanisms that (amongst others) enable bacterial cells to enter the persister state. The Phd/Doc toxin-antitoxin system is a representative of a distinct family of toxin-antitoxin modules found in bacteria. The aim of the work was to determine the DNA specificity and biophysical properties of Phd, the antitoxin from the Phd/Doc toxin-antitoxin module, using isothermal titration calorimetry and an assortment of other methods. The first steps in the project were the purification of Phd from an E. coli cell lysate using an on-column unfolding/refolding procedure. Next, the suitability of the purified Phd was assessed using circular dichroism spectroscopy, and the effect of buffer composition on the thermal stability of the protein was assessed with a thermal stability assay. The specificity of Phd for its DNA operator was measured using isothermal titration calorimetry. The 8 base pair palindromic sequence was mutated to help determine which bases affect the specificity the most. The secondary structure of the isolated protein was found to be in accordance with a published reference crystal structure. Determining the thermal stability of Phd in buffers of varying composition was inconclusive, as the intrinsically disordered nature of the N-terminal protein domain interferes with the dye used in the stability assay. The data obtained by isothermal titration calorimetry enabled a limited glimpse into the operator specificity of Phd. All of the investigated mutants have significantly worse binding properties as the wild type oligonucleotide.

• Keywords: proteins, toxin-antitoxin, isothermal titration calorimetry, thermodynamics, DNA interactions