Abstract |
Small heat shock proteins (sHSPs) and the related alpha-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/ deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95-156 and 228-252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.
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Authors | Guilong Cheng, Eman Basha, Vicki H Wysocki, Elizabeth Vierling |
Journal | The Journal of biological chemistry
(J Biol Chem)
Vol. 283
Issue 39
Pg. 26634-42
(Sep 26 2008)
ISSN: 0021-9258 [Print] United States |
PMID | 18621732
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Chemical References |
- Chaperonin 60
- Escherichia coli Proteins
- HSP16.9 protein, Triticum aestivum
- HSP18 protein, plant
- HSP70 Heat-Shock Proteins
- Heat-Shock Proteins
- Multiprotein Complexes
- Plant Proteins
- Recombinant Proteins
- Deuterium
- Malate Dehydrogenase
- dnaK protein, E coli
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Topics |
- Animals
- Chaperonin 60
(chemistry, metabolism)
- Deuterium
(chemistry, metabolism)
- Deuterium Exchange Measurement
(methods)
- Escherichia coli Proteins
(chemistry, metabolism)
- HSP70 Heat-Shock Proteins
(chemistry, metabolism)
- Heat-Shock Proteins
(chemistry, metabolism)
- Malate Dehydrogenase
(chemistry, metabolism)
- Mass Spectrometry
(methods)
- Multiprotein Complexes
(chemistry, metabolism)
- Peas
(chemistry)
- Plant Proteins
(chemistry, metabolism)
- Protein Folding
- Recombinant Proteins
(chemistry, metabolism)
- Swine
- Triticum
(chemistry)
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