Thermodynamic Studies on the Cataract-Associated Mutant, E107a, of Human Gamma-D Crystallin: Molecular Basis for Cataract Formation

Abstract

The E107A mutation in human gammaD-crystallin (HGD) is associated with nuclear cataract. The concentration of crystallins in the lens is extremely high, approaching that within protein crystals. The composition in the lens nucleus, i.e. the central core, is such that the alpha- and gamma crystallin weight fractions are comparable. In earlier studies we showed that mutations in HGD dramatically compromised protein solubility, while maintaining the overall protein structure. In contrast, in the E107A mutation neither the structure (circular dichroism and tryptophan fluorescence emission) and stability (thermal denaturation), nor the solubility are affected significantly. However, as expected, the mutation raises the pI by ∼1 pH unit, i.e. from 7.4 to 8.4. The increase in pI suggested a change in the interaction of E107A with alpha crystallin, which is negatively charged at neutral pH. To determine changes in such interaction, we compared the liquid-liquid phase boundaries in binary mixtures containing either HGD or E107A, and alpha crystallin. Our preliminary studies show that while the phase-separation temperatures of mixtures in the two cases (i.e. HGD+alpha and E107A+alpha) remain comparable, the nature of the paired compositions of the two phases in equilibrium are distinct. In particular, the tie-line slopes are altered in the direction predicted to correspond to increased alpha-gamma attraction, on the basis of molecular dynamics simulation and thermodynamic perturbation theory (1). Thus, it appears that increased attractive interactions between the E107A mutant of HGD and alpha crystallin could destabilize the crystallin mixture in the lens nucleus, and lead to increased light scattering and cataract.(1) N. Dorsaz, G. M. Thurston, A. Stradner, P. Schurtenberger and G. Foffi, J. Phys. Chem. B 113:1693-1709 (2009)Supported by: NIH Grant EY 18249-01 (G.T.) and EY 10535 (J.P.)