f7: Thermal stability of wild type and G98R αA-crystallin upon Cu2+-binding. Aggregation of 0.2 mg/ml of αA-crystallin (curve 1) and G98R αA-crystallin (curve 2) in buffer B and of 30 μM Cu2+-treated samples of αA-crystallin (curve 3) and G98R αA-crystallin (curve 4) is shown. The aggregation was monitored by light scattering at 465 nm as a function of temperature. G98R mutation in αA-crystallin leads to decreased thermal stability upon Cu2+-binding.

Mentions: α-crystallins in general are highly thermostable with respect to large unfolding of their secondary structural contents [5,40,41]. However, they show a transition around 60 °C exhibiting hydrophobicity changes [5,40-42]. Therefore, we have studied the thermostability by monitoring light scattering. αA-crystallin exhibits a sharp (cooperative) transition in light scattering around 66 °C (Figure 7). In the presence of Cu2+, the light scattering profile of αA-crystallin exhibits a gradual increase till about 68 °C and exhibits a sharp transition with an inflection point around 76 °C, indicating that Cu2+-binding stabilizes αA-crystallin against heat-induced self-aggregation. In conformity with our earlier observations [32,33], the light scattering of G98R αA-crystallin increases above 50 °C in a less cooperative manner (Figure 7). Interestingly, the light scattering profile of the Cu2+-bound G98R αA-crystallin increases around 35 °C, which is more pronounced above 55 °C. Thus, Cu2+-binding further destabilizes G98R αA-crystallin against heat-induced aggregation.

Singh D, Tangirala R, Bakthisaran R, Chintalagiri MR - Mol. Vis. (2009)

Bottom Line: Interestingly, Cu(2+) binding increases the chaperone-like activity of alphaA-crystallin toward the aggregation of citrate synthase at 43 degrees C while it decreases the chaperone-like activity of G98R alphaA-crystallin.Mixed oligomer formation between the wild type and the mutant subunits modulates the Cu(2+)-induced effect on the self-aggregation propensity.Other heavy metal ions, namely Cd(2+) and Zn(2+) but not Ca(2+), also promote the self-aggregation of G98R alphaA-crystallin and decrease its chaperone-like activity.Our study demonstrates that unlike wild type alphaA-crystallin, G98R alphaA-crystallin and its mixed oligomers with wild type protein are vulnerable to heavy metal ions.

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.

Abstract: alphaA- and alphaB-crystallins are abundantly present in the eye lens, belong to the small heat shock protein family, and exhibit molecular chaperone activity. They are also known to interact with metal ions such as Cu(2+), and their metal-binding modulates the structure and chaperone function. Unlike other point mutations in alphaA-crystallin that cause congenital cataracts, the G98R mutation causes pre-senile cataract. We have investigated the effect of Cu(2+) on the structure and function of G98R alphaA-crystallin.Fluorescence spectroscopy and isothermal titration calorimetry were used to study Cu(2+) binding to alphaA- and G98R alphaA-crystallin. Circular dichroism spectroscopy was used to study secondary and tertiary structures, and dynamic light scattering was used to determine the hydrodynamic radii of the proteins. Chaperone activity and self-aggregation of the wild type and the mutant protein in the absence and the presence of the metal ions was monitored using light scattering.Our fluorescence quenching and isothermal titration calorimetric studies show that like alphaA-crystallin, G98R alphaA-crystallin binds Cu(2+) with picomolar range affinity. Further, both wild type and mutant alphaA-crystallin inhibit Cu(2+)-induced generation of reactive oxygen species with similar efficiency. However, G98R alphaA-crystallin undergoes pronounced self-aggregation above a certain concentration of Cu(2+) (above subunit to Cu(2+) molar ratio of 1:3 in HEPES-NaOH buffer, pH 7.4). At concentrations of Cu(2+) below this ratio, G98R alphaA-crystallin is more susceptible to Cu(2+)-induced tertiary and quaternary structural changes than alphaA-crystallin. Interestingly, Cu(2+) binding increases the chaperone-like activity of alphaA-crystallin toward the aggregation of citrate synthase at 43 degrees C while it decreases the chaperone-like activity of G98R alphaA-crystallin. Mixed oligomer formation between the wild type and the mutant subunits modulates the Cu(2+)-induced effect on the self-aggregation propensity. Other heavy metal ions, namely Cd(2+) and Zn(2+) but not Ca(2+), also promote the self-aggregation of G98R alphaA-crystallin and decrease its chaperone-like activity.Our study demonstrates that unlike wild type alphaA-crystallin, G98R alphaA-crystallin and its mixed oligomers with wild type protein are vulnerable to heavy metal ions. Our study provides insight into aspects of how environmental factors could augment phenotype(s) in certain genetically predisposed conditions.