Abstract
Purpose: :
To understand the molecular phenotypes associated with functional defects in the mutant γC–crystallin molecule due to a 5–bp insertion and an R168W mutation seen in congenital zonular pulverulant and congenital lamellar cataract, respectively.
Methods: :
We have cloned, expressed, isolated and compared the solution state structural features of the mutants with those of normal (wild type) γC–crystallin. Normal and mutant γ–crystallin genes were cloned into pET21a(+)ve and pGEX 5x–3 vector and were over–expressed in BL–21(DE3)pLysS strain of E.coli. The recombinant proteins were isolated and purified using ion–exchange and molecular sieving chromatography. Structural characterization of the mutant and normal proteins was done by molecular spectroscopic techniques such as circular dichroism and fluorescence, while structural stability was assessed by thermal and chemical denaturation, self association of the proteins was studied using temperature dependent light scattering.
Results: :
We find that the 5–base insertion led to a loss of secondary and tertiary structures of the molecule, and to an enhanced tendency of self–aggregation into light–scattering particles, offering a possible factor in lens opacification. The R168W mutant, on the other hand, is remarkably similar to the wild type molecule in its conformation and structural stability, but differ in its quaternary structure, or ability to aggregate and scatter light.
Conclusions: :
It thus appears that unfolding or structural destabilization is not always necessary for crystallin–associated cataractogenesis. Quaternary structural differences suffice to enhance lens clouding.
Keywords: protein structure/function • protein purification and characterization • cataract