Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. βB2 is the most abundant β-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. βB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among β-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type βB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes βB2 and may disrupt the function of βB2 in the lens.
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