Project Summary Age-related cataract is associated with extensive deamidation, racemization, and isomerization of crystallins, the major refractive proteins of the lens. Extensive studies by our laboratories and others have found deamidation significantly decreases the stability of crystallins with minimal structural perturbations. However, these deamidation mimics created using mutagenesis did not readily aggregate in vitro. We hypothesize that this is because they were lacking racemization and isomerization, two modifications accompanying deamidation that may be more disruptive to crystallin structure than deamidation alone. Unlike deamidation, these modifications cannot be introduced into crystallins by genetic means. This has prevented studies to gauge the importance of these additional modifications in age-related cataract. Therefore, the purpose of these experiments is to create γS-crystallins containing specific sites of physiologically relevant racemized and isomerized aspartates that result from the age-related deamidation process and determine their effect on protein structure. The specific aims are to: 1) use synthetic heavy peptide standards and high-resolution mass spectrometry to determine the relative proportion of different racemized and isomerized aspartates in γS- crystallin from the insoluble protein of aged cataractous human lenses so that relevant species can be selected, 2) introduce these racemized and isomerized residues into γS-crystallin using semi-synthetic processes that have never before been used in the lens field, and 3) examine the result of these modifications on γS-crystallin structure using protein stability and light scattering measurements, hydrogen/deuterium exchange mass spectrometry, and nuclear magnetic resonance spectroscopy. These experiments will, for the first time, critically test the potential impact of age-related racemization and isomerization on crystallin structure. These results could play an important role in slowing cataract development by developing drugs that specifically prevent the aggregation and light scatter of racemized and isomerized crystallins in aged lenses.