Structure and Dynamics of the Fish Eye Lens Protein, γM7-Crystallin

Abstract

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.

NMR Studies of Eye Lens Crystallins

“…Therefore, the lens crystallins of aquatic organisms proteins must have higher refractive index increments, exist at higher concentrations, or both. The structure of the zebrafish γ M7-crystallin has been solved, revealing the same overall fold as that of the mammalian γ -crystallins 64 ; however, important details differ, as expected. γ M7-crystallin has enhanced methionine content and lacks some of the tryptophan residues that are very strongly conserved among mammals, and, thus, the loop encompassing residues 65-72 in the N-terminal domain is less tightly packed against the hydrophobic core than in the homologous mammalian proteins.…”

Functions of crystallins in and out of lens: Roles in elongated and post-mitotic cells

“…In contrast, the major human γ-crystallins of the denser lens core, γC- and γD-crystallins, have Arg/Lys ratios of 20/3 and 21/1, respectively, while in Danio rerio γM2b- and γM7-crystallins the ratios are 21/3 and 20/2 respectively. The extremely high RI in the core of fish lenses may also benefit from γM-crystallins having very large numbers of methionines on the surface (Chen et al, 2013; Mahler et al, 2013; Zhao et al, 2013). The presence of these residues is associated with extremes in the hydrodynamic properties of compactness and low frictional ratio which are common to all γ-crystallins but particularly prominent in γM-crystallins of fish lenses.…”

“…These make important contributions to the hydrophobic core of each domain and to domain stability (Mahler et al, 2013). Tryptophan side chains can absorb and emit photons in the UV range.…”

The βγ-crystallins: Native state stability and pathways to aggregation

“…The cataract-linked A2V mutation in the βB2 crystallin represents another interesting case: while virtually identical to the wild-type protein in thermodynamic stability and aggregation propensity, this mutant was found to be impaired in higher-order oligomeric assembly (Xu et al, 2012). Recent work on the fish γM-crystallins (Mahler et al, 2013) provides a particularly striking example. The fish crystallins exist in conditions of extreme crowding, reaching concentrations of up to 1 g/ml.…”

“…Thus, the fish γM-crystallins lack the protective four-tryptophan cluster of their mammalian counterparts, presumably due to their reduced need for protection from UV exposure (Mahler et al, 2013). Some types of damage – such as from occupational exposure to heavy metals – may not have been major factors during the evolution of the human lens, but are more significant today.…”