Title | Thermodynamic Stability of the Transcription Regulator PaaR2 from Escherichia coli O157:H7. |
Publication Type | Journal Article |
Year of Publication | 2019 |
Authors | De Bruyn, P., S. Hadži, A. Vandervelde, A. Konijnenberg, M. Prolič-Kalinšek, Y. G. - J. Sterckx, F. Sobott, J. Lah, L. Van Melderen, and R. Loris |
Journal | Biophys J |
Volume | 116 |
Issue | 8 |
Pagination | 1420-1431 |
Date Published | 2019 Apr 23 |
ISSN | 1542-0086 |
Abstract | 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. |
DOI | 10.1016/j.bpj.2019.03.015 |
Alternate Journal | Biophys. J. |
PubMed ID | 30979547 |
PubMed Central ID | PMC6486494 |
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