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 gamma D Crystallin (H gamma D-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S H gamma D-Crys and 190F H gamma D-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 H gamma D-Crys and L5S H gamma D-Crys by 0.3 M GuHCl, the C-terminal domain (C-td) by 0.6 M GuHCl, and the single transition of 190F H gamma D-Crys by 0.4 M GuHCl. In kinetic unfolding reactions, sodium citrate stabilization effect was observed only for the mutant 190F H gamma D-Crys. In the presence of citrate, a kinetic unfolding intermediate of 190F H gamma D-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 H gamma D-Crys, but considerably slowed the rate of aggregation of both L5S H gamma D-Crys and 190F H gamma D-Crys. The presence of sodium citrate dramatically slowed refolding of WT H gamma D-Crys and 190F H gamma D-Crys, but had a significantly smaller effect on the refolding of L5S H gamma D-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 solutionbased 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. Published by Elsevier Ltd.