Thermodynamic stability of the transcription regulator PaaR2 from Escherichia coli O157:H7

Pieter De Bruyn, San Hadzi, Alexandra Vandervelde, Albert Konijnenberg, Maruša Prolič Kalinšek, Yann Sterckx, Frank Sobott, Jurij Lah, Laurence Van Melderen, Remy Loris

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4 Citations (Scopus)


PaaR2 is a putative transcription regulator encoded by a three-component parDE-like toxin-antitoxin module from Escherichia coli O157:H7. Although this module's toxin, antitoxin, and toxin-antitoxin complex have been more thoroughly investigated, little remains known about its transcription regulator PaaR2. Using a wide range of biophysical techniques (circular dichroism spectroscopy, size-exclusion chromatography-multiangle laser light scattering, dynamic light scattering, small-angle x-ray scattering, and native mass spectrometry), we demonstrate that PaaR2 mainly consists of α-helices and displays a concentration-dependent octameric build-up in solution and that this octamer contains a global shape that is significantly nonspherical. Thermal unfolding of PaaR2 is reversible and displays several transitions, suggesting a complex unfolding mechanism. The unfolding data obtained from spectroscopic and calorimetric methods were combined into a unifying thermodynamic model, which suggests a five-state unfolding trajectory. Furthermore, the model allows the calculation of a stability phase diagram, which shows that, under physiological conditions, PaaR2 mainly exists as a dimer that can swiftly oligomerize into an octamer depending on local protein concentrations. These findings, based on a thorough biophysical and thermodynamic analysis of PaaR2, may provide important insights into biological function such as DNA binding and transcriptional regulation.

Original languageEnglish
Pages (from-to)1420-1431
Number of pages12
JournalBiophysical Journal
Issue number8
Publication statusPublished - 23 Apr 2019

Bibliographical note

Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.


  • Toxin-antitoxin module
  • Transcription factors
  • Molecular biophysics
  • Protein chemistry


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