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

Journal Contribution - Journal Article

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.

Journal: Biophysical Journal
ISSN: 0006-3495
Issue: 8
Volume: 116
Pages: 1420-1431
Number of pages: 12
Publication year:2019
Keywords:Toxin-antitoxin module, Transcription factors, Molecular biophysics, Protein chemistry, Biochemistry/biophysics/molecular biology
  • ORCID: /0000-0003-4175-1858/work/86797584
  • ORCID: /0000-0001-7126-6103/work/82915128
  • DOI: https://doi.org/10.1016/j.bpj.2019.03.015
  • Scopus Id: 85063982820
  • PubMed Id: 30979547
  • WoS Id: 000465230800006
  • PubMed Id: PMC6486494
Accessibility:Open