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S.L. Flaugh, I.A. Mills, J. King; The Effects of Deamidation on the Stability and Aggregation of Human D Crystallin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3488.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Human γD–crystallin (HγD–Crys) is a primarily ß–sheet, two domain lens crystallin found in the protein aggregates associated with mature–onset cataract. The two domains of HγD–Crys interact through inter–domain side chain contacts including a pairing of two glutamine residues, Gln54 and Gln143. The crystallin proteins do not turnover in mature lens cells and thus must remain stable for decades despite the continued presence of environmental insults. These stresses result in covalent damage to the crystallins, including glutamine and asparagine deamidation. Covalent damage may cause partial unfolding into aggregation–prone conformations that are precursors to cataract. We have analyzed the effects of deamidation of Gln54 and Gln143 on the stability, folding and aggregation of HγD–Crys. Methods: Single and double glutamine to glutamate substitution mutants of Gln54 and Gln143 were constructed, expressed, and the mutant proteins purified. Equilibrium unfolding/refolding experiments of the purified wild type and mutant proteins were performed in GuHCl at pH 7.0 or Urea at pH 2.0. Aggregation was monitored by solution turbidity measurements. Results: All deamidation mutants were destabilized compared to wild type at pH 7.0. The mutants populated a partially unfolded intermediate at 2.3 M GuHCl that likely has a folded C–terminal domain and unfolded N–terminal domain. When the pH was decreased to 3.0, equilibrium unfolding/refolding transitions of the mutant and wild type proteins were identical. In contrast, alanine substitution of these residues destabilized HγD–Crys at both pH 2.0 and 7.0. Diluting the partially folded intermediate out of denaturant resulted in the accumulation of a high molecular aggregate that scattered light. Conclusions: These results indicate that deamidation of Gln54 and/or Gln143 destabilizes HγD–Crys into a single–folded domain conformer. This destabilization is the result of introducing a negative charge into a partially–buried region of the domain interface. The aggregation reaction that proceeds from the partially folded conformer may mimic a reaction significant in mature–onset cataract.
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