Purchase this article with an account.
T. Takata, K. Lampi; Deamidation at the Dimer Interface Destabilizes betaA3-Crystallin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4424.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Previous studies have suggested deamidation destabilizes betaB1 and betaB2-crystallins. During normal aging, deamidation is one of the most prevalent modifications of betaA3-crystallin. The purpose of this study was to determine the effects of two potential deamidation sites across the interface of the betaA3-crystallin dimer.
Glutamic acid substitutions at the previously reported in vivo site at Q180 in the C-terminal domain, at the homologous site Q85 in the N-terminal domain, and the double mutation (Q85E/Q180E) were constructed by site-directed mutagenesis. The structure and stability of the proteins were characterized by the biophysical measurements of circular dichroism, multiangle light scattering, and fluorescence during unfolding in urea.
The mutants had a native-like secondary structure by circular dichroism spectroscopy similar to betaA3-crystallin wild type, with differences in tertiary structure that were greater for the double mutant. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation and size were not disrupted. The equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition for the single and double mutants suggesting an intermediate. Deamidation also increased the susceptibility to enzymatic digestion.
These results suggest deamidation at the interface increase exposure of internal residues, contributing to the lowered stability without dissociating the dimer, similar to our previously reported findings for betaB2-crystallin. These in vitro results suggest that deamidation at a known in vivo site during aging may be disruptive to the function of betaA3 in the lens.
This PDF is available to Subscribers Only