Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry.

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.

• 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)

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

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)