A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in γS-crystallin

Soojin Lee, Bryon Mahler, Jodie Toward, Blake Jones, Keith Wyatt, Lijin Dong, Graeme Wistow, Zhengrong Wu

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases γS-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse γS-crystallin causes the severe Opj cataract, with disruption of cellular organization and appearance of fibrillar structures in the lens. Although the mutant protein has a near-native fold at room temperature, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected γ-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to a 3.5-kcal/mol reduction in the free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At a higher temperature, this effect further increases to allow for a much more uniform exchange rate among the N-terminal core residues and those of the least well-structured surface loops. This suggests a concerted unfolding intermediate of the N-terminal domain, while the C-terminal domain stays intact. Increasing concentrations of guanidinium chloride produced two transitions for the Opj mutant, with an unfolding intermediate at γ 1 M guanidinium chloride. The consequence of this partial unfolding, whether by elevated temperature or by denaturant, is the formation of thioflavin T staining aggregates, which demonstrated fibril-like morphology by atomic force microscopy. Seeding with the already unfolded protein enhanced the formation of fibrils. The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of aggregates with some of the characteristics of amyloid fibrils.

Original languageEnglish (US)
Pages (from-to)320-330
Number of pages11
JournalJournal of Molecular Biology
Volume399
Issue number2
DOIs
StatePublished - Jun 4 2010
Externally publishedYes

Fingerprint

Crystallins
Mutant Proteins
Guanidine
Mutation
Temperature
Protein Unfolding
Crystalline Lens
Atomic Force Microscopy
Deuterium
Amyloid
Cataract
Lenses
Vertebrates
Hydrogen
Staining and Labeling
Proteins

Keywords

  • γS-crystallin
  • Amyloid
  • Cataract
  • Denaturation
  • H/D exchange

ASJC Scopus subject areas

  • Molecular Biology

Cite this

A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in γS-crystallin. / Lee, Soojin; Mahler, Bryon; Toward, Jodie; Jones, Blake; Wyatt, Keith; Dong, Lijin; Wistow, Graeme; Wu, Zhengrong.

In: Journal of Molecular Biology, Vol. 399, No. 2, 04.06.2010, p. 320-330.

Research output: Contribution to journalArticle

Lee, Soojin ; Mahler, Bryon ; Toward, Jodie ; Jones, Blake ; Wyatt, Keith ; Dong, Lijin ; Wistow, Graeme ; Wu, Zhengrong. / A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in γS-crystallin. In: Journal of Molecular Biology. 2010 ; Vol. 399, No. 2. pp. 320-330.
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