Characterization of the unfolding, refolding, and aggregation pathways of two protein implicated in cataractogenesis : human gamma D and human gamma S crystallin

Abstract

Human [gamma]D crystallin (H[gamma]D-Crys) and human [gamma]S crystallin (H[gamma]S-Crys), are major proteins of the human eye lens and are components of cataracts. H[gamma]D-Crys is expressed early in life in the lens cortex while H[gamma]S-Crys is expressed throughout life in the lens epithelial cells. Both are primarily β-sheet proteins made up of four Greek keys separated into two domains and display 69% sequence similarity. The unfolding and refolding of H[gamma]D-Crys and H[gamma]S-Crys have been characterized as a function of guanidinium hydrochloride (GdnHCl) concentration at neutral pH and 37⁰C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Equilibrium unfolding and refolding experiments with GdnHCl showed unfolded protein is more fluorescent than its native counter-part despite the absence of metal or ion-tryptophan interactions in both of these proteins. This fluorescence quenching may influence the lens response to ultraviolet light radiation or the protection of the retina from ambient ultraviolet damage. Wild-type H[gamma]D-Crys exhibited reversible refolding above 1.0 M GdnHCl. Aggregation of refolding intermediates of H[gamma]D-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GdnHCl, beyond the major conformational transition region. H[gamma]S-Crys, however, exhibited a two-state reversible unfolding and refolding with no evidence of aggregation. Atomic force microscopy of H[gamma]D-Crys samples under aggregating conditions revealed ordered fiber structures that could recruit H[gamma]S-Crys to the aggregate. To provide fluorescence reporters

(cont.) for each quadrant of H[gamma]D-Crys, triple mutants each containing three tryptophan to phenylalanine substitutions and one native tryptophan have been constructed and expressed. Trp68-only and Trp 156-only retained the quenching pattern of wild-type H[gamma]D-Crys. During equilibrium refolding/unfolding, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple mutant tryptophan proteins identified an intermediate along the H[gamma]D-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of H[gamma]D-Crys. An N143D deamination post-translational modification has recently been identified in H[gamma]S-Crys that is present in high concentrations in insoluble protein removed from cataractous lenses. The presence of the N143D mutation did not significantly affect the equilibrium or kinetic properties of H[gamma]S-Crys indicating that this mutation is unlikely to be involved in protein destabilization during cataract formation in vivo. The method in which H[gamma]D-Crys aggregates on its own and engages neighboring molecules in the polymerization process in vitro may provide insight into the process of cataractogenesis in vivo.