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J Mol Biol. 2018 Jan 5;430(1):33-40. doi: 10.1016/j.jmb.2017.11.002. Epub 2017 Nov 11.

The Mechanism of HdeA Unfolding and Chaperone Activation.

Author information

1
Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland. Electronic address: loic.salmon@mol.biol.ethz.ch.
2
Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
3
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
4
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
5
Institute für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
6
Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO 80045, USA.
7
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA; Department of Chemistry, Duke University, Durham, NC 27708, USA.
8
Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address: jbardwel@umich.edu.
9
Department of Chemistry & Biochemistry and the Knoebel Institute for Healthy Aging, University of Denver, Denver, CO 80208, USA. Electronic address: scott.horowitz@du.edu.

Abstract

HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition from folded dimer to unfolded monomer between pH3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.

KEYWORDS:

NMR; acid; chaperone; protein folding

PMID:
29138002
PMCID:
PMC5738273
DOI:
10.1016/j.jmb.2017.11.002
[Indexed for MEDLINE]
Free PMC Article

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