Abstract
Purpose: :
Conformational change and aggregation of native proteins is associated with many serious age-related and neurological diseases. γS-crystallin is a highly stable, abundant structural component of the 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 the appearance of fibrillar structures in the lens.
Methods: :
Multi-dimensional nuclear magnetic resonance (NMR) and several bio-physical methods such as atomic force microscopy (AFM) were employed to study the destabilization effect by F9S mutation.
Results: :
Although at room temperature the mutant protein has a near native fold, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected β-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to 3.5 kcal/mol reduction in free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At higher temperature, this effect further increases to allow 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 HCl produced two transitions for the Opj mutant, with an unfolding intermediate at ~1M GuHCl. The consequence of this partial unfolding, whether by elevated temperature or denaturant, is the formation of Thioflavin T staining aggregates, which demonstrated fibril-like morphology by AFM. Seeding with already unfolded protein enhanced formation of amyloid-like fibrils. Congo red staining of Opj lens shows fairly large clumps of intensely fluorescent material in cortical fibers, reminiscent of the fibrils in the Opj cataractous lenses by EM images.
Conclusions: :
The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of amyloid-like fibrillar aggregates.
Keywords: cataract • crystallins