Citrate Binding Stabilizes Human Gamma-Crystallin to Slow Unfolding and Inhibit Aggregation

Abstract

Recent studies have demonstrated that small molecules can bind destabilized or aggregation prone proteins to prevent unfolding and aggregation. Cataract, the leading cause of blindness worldwide, is caused by aggregation of proteins in the eye lens. The most abundant proteins in the eye lens belongs to the βγ-Crystallin superfamily, which accounts for 90% of lens protein composition. These proteins are synthesized in utero and must remain stable and soluble throughout life. Damage, or deleterious post-translational modification can destabilize these proteins and induce aggregation-prone conformations. Sodium citrate has been shown to prevent unfolding and aggregation of alpha-antitrypsin by stabilizing secondary structure. In this study, we demonstrate the effects of citrate binding on both wild-type and disease models of Human γD Crystallin. Equilibrium unfolding-refolding experiments show an increase in the ΔG of unfolding with increasing concentrations of sodium citrate, while kinetic experiments show that sodium citrate slows the rate of unfolding in denaturant. UV resonance Raman spectroscopy has been used to examine Trp residues in the protein and monitor vibrational modes as a function of temperature. Preliminary results indicate a resistance to unfolding in the presence of citrate. The effect does not appear to be due to metal ion chelation, and may reflect direct binding to the crystallins, as with anti-trypsin.