Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry.
J Biol Chem
; 283(39): 26634-42, 2008 Sep 26.
Article
em En
| MEDLINE
| ID: mdl-18621732
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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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Plantas
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Dobramento de Proteína
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Complexos Multiproteicos
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Deutério
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Proteínas de Choque Térmico
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Malato Desidrogenase
Limite:
Animals
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article