title: A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in gammaS-crystallin.
abstract: Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases. gammaS-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse gammaS-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 beta-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 approximately 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.
authors.affiliation: Biochemistry Department, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Soojin
authors.initials: S
authors.lastname: Lee
authors.forename: Bryon
authors.initials: B
authors.lastname: Mahler
authors.forename: Jodie
authors.initials: J
authors.lastname: Toward
authors.forename: Blake
authors.initials: B
authors.lastname: Jones
authors.forename: Keith
authors.initials: K
authors.lastname: Wyatt
authors.forename: Lijin
authors.initials: L
authors.lastname: Dong
authors.forename: Graeme
authors.initials: G
authors.lastname: Wistow
authors.forename: Zhengrong
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000818:Animals; D051379:Mice; D008958:Models, Molecular; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D013696:Temperature; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics*
chemical_list: D038222:gamma-Crystallins
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