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.Cataract formation is the leading cause of blindness worldwide and affects an estimated 16 to 20 million people (1). Crystallins are highly soluble structural proteins that comprise 90% of lens proteins (2). The highly ordered, tightly packed crystallins make up the transparent structure of the lens and allow it to focus light onto the retina. These proteins undergo little turnover during the human life span, allowing for accumulation of modifications that may decrease crystallin solubility, alter lens transparency, and diminish vision.During normal aging, crystallins are modified by truncation, oxidation, deamidation, and disulfide bond formation (3-9). Most notable by two-dimensional gel electrophoresis are trains of deamidated species (3). Each of the major crystallin polypeptides in the lens is deamidated during aging, and most are deamidated at multiple residues. Deamidation introduces a negative charge at physiological pH by replacing an amide with a carboxyl group and causes some isomerization. These changes potentially disrupt protein structure.Recently, 34 sites of deamidations have been identified in a single cataractous lens (10). Due to the difficulty of detecting a single mass unit change resulting from deamidation, quantitative data associating deamidation with cataract formation is limited. The large numbers of † Funding provided by the National Institutes of Health, National Eye Institute, Grant Reference No. R01-EY012239 (K.J.L.), Oregon Lions Sight and Hearing Foundation grant (K.J.L.), Shriners Hospitals Shared Facilities Grant, a...