Inhibition of unfolding and aggregation of lens protein human gamma D crystallin by sodium citrate

Cataract affects 1 in 6 Americans over the age of 40, and represents a global health problem. Mature onset cataract is associated with the aggregation of partially unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the primary effective treatment for cataract. As an alternative preventive approach, small molecules have been suggested as potential therapeutic agents. In this work, we study the effect of sodium citrate on the stability of Human γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding–refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-terminal domain (N-td) of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-terminal domain (C-td) by 0.6 M GuHCl, and the single transition of I90F HγD-Crys by 0.4 M GuHCl. In kinetic unfolding reactions, sodium citrate stabilization effect was observed only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-Crys was observed, which was not populated in the absence of citrate. The rates of aggregation were measured using solution turbidity. Sodium citrate demonstrated negligible effect on rate of aggregation of WT HγD-Crys, but considerably slowed the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slowed refolding of WT HγD-Crys and I90F HγD-Crys, but had a significantly smaller effect on the refolding of L5S HγD-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results indicate that assessment of potential anti-cataract agents needs to include effects on the unfolding and aggregation pathways, as well as the native state.