Characterization of DNA-specificity and thermal stability of the Phd protein

  • Jaka Kranjc ((PhD) Student)
  • Remy Loris (Co-promotor)
  • Janez Ilaš (Promotor)

Student thesis: Master's Thesis

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.
Date of Award5 May 2020
Original languageEnglish
Awarding Institution
  • University of Ljubljana
SponsorsEC, Erasmus+
SupervisorRemy Loris (Co-promotor), Janez Ilaš (Promotor), Simon Žakelj (Jury), Ariel Talavera Perez (Advisor), Gopinath Muruganandam (Advisor) & Sarah Haesaerts (Advisor)

Keywords

  • toxin-antitoxin module
  • thermodynamics
  • Isothermal titration calorimetry
  • protein-DNA interactions

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