A Single Destabilizing Mutation (F9S) Promotes Concerted Unfolding of an Entire Globular Domain in γS-Crystallin

Abstract

Conformational change and aggregation of native proteins are associated with many serious agerelated 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 β-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 ~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.

The Human W42R γD-Crystallin Mutant Structure Provides a Link between Congenital and Age-related Cataracts*

“…At 37°C, a noticeable amount (ϳ10%) of partially folded species is present, similar to related observations for a F9S mutant of mouse ␥S-crystallin that is associated with Opj cataract in mice and for which an unfolding intermediate was found to be populated to ϳ1-2% at physiological conditions (19,20).…”

“…For other crystallin mutants such as murine ␥S-F9S (9) and human ␥C-T5P (18), a transient unfolding intermediate was observed under physiological conditions (19). R 2 relaxation dispersion experiments on the murine ␥S-F9S mutant showed an ϳ2% population of a loosely structured intermediate at 37°C that increased with temperature (20).…”

Wild-type Human γD-crystallin Promotes Aggregation of Its Oxidation-mimicking, Misfolding-prone W42Q Mutant

“…Intrinsic fluorescence showed native-like structure at 37°C but a prominent unfolding intermediate under partially denaturing conditions (Fig. 2C), similar to other cataract-linked ␥-crystallin core substitutions (8,(21)(22)(23). The I 360 /I 320 ratio of the N-unfolded intermediate was ϳ0.7, because the W42Q mutant is missing one of the two Trp residues in the N-terminal domain.…”

“…More than 20 point mutations in conserved ␥-crystallin residues, particularly near the N-terminal ␤-strands, are linked to congenital cataract (14). Such mutations can generate conformational intermediates leading to aggregation (8,(21)(22)(23). The hydrophobic core of each double-Greek key domain of H␥D includes two highly conserved Trp residues ( Fig.…”

NMR Studies of Eye Lens Crystallins

“…The structure of the F9S variant was also solved using MFR methodology and was found to have the same double-Greek key domain fold as wild-type γ S and related γ -crystallins. 61 Guanidinium chloride (GuCl) denaturation of the variant resulted in a three-state unfolding with transitions at 0.5 and 2.3 M GuCl, in which an intermediate is produced wherein the C-terminal domain is folded and the Nterminal domain is unfolded. By comparison, wild-type γ S has a monophasic unfolding curve that takes place at 2.5 M GuCl, consistent with the previous finding that the two domains of γ S-crystallin stabilize each other 62 -this extra stabilization is lost in F9S because the N-terminal domain unfolds first.…”