The vertebrate eye lens contains high concentrations of crystallins. The dense lenses of fish are particularly abundant in a class called γM-crystallin whose members are characterized by an unusually high methionine content and partial loss of the four tryptophan residues conserved in all γ-crystallins from mammals which are proposed to contribute to protection from UV-damage. Here, we present the structure and dynamics of γM7-crystallin from zebrafish (Danio rerio). The solution structure shares the typical two-domain, four-Greek-key motif arrangement of other γ-crystallins, with the major difference noted in the final loop of the N-terminal domain, spanning residues 65–72. This is likely due to the absence of the conserved tryptophans. Many of the methionine residues are exposed on the surface but are mostly well-ordered and frequently have contacts with aromatic side chains. This may contribute to the specialized surface properties of these proteins that exist under high molecular crowding in the fish lens. NMR relaxation data show increased backbone conformational motions in the loop regions of γM7 compared to those of mouse γS-crystallin and show that fast internal motion of the interdomain linker in γ-crystallins correlates with linker length. Unfolding studies monitored by tryptophan fluorescence confirm results from mutant mouse γS-crystallin and show that unfolding of a βγ-crystallin domain likely starts from unfolding of the variable loop containing the more fluorescently quenched tryptophan residue, resulting in a native-like unfolding intermediate.
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.
In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows adoption of a native fold but renders the protein susceptible to temperature and concentration-dependent aggregation, including fibril formation. Hydrogen/Deuterium exchange and denaturant unfolding studies on this mutant (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here we have used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea Opj forms a stable and partially unfolded entity, characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill (CPMG) T2-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold may explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this also shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light scattering centers in the cataractous lens.
Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM–focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.
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