The crystallins, which maintain the transparency of the eye lens by providing a well-defined gradient of refractive index, are a fascinating and medically relevant group of proteins. In contrast to most other proteins, which are constantly subject to degradation, these proteins have very low turnover and must remain intact for a lifetime. This is even more remarkable considering their extremely high concentration in the lens (more than 400 mg/mL). The major types of crystallins can be categorized as either structural (???) or solubilizing (?). Cataract, a major cause of blindness worldwide, results when the structural crystallins aggregate or phase- separate, leading to opacity of the lens. Over time, degradation of the crystallins occurs when the crystallins become chemically modified, often by deamidation or truncation when damaged by UV light, or by glycation in the case of diabetes. In addition to age-related cataracts, several known point mutations cause hereditary juvenile-onset cataracts. Despite the medical and biophysical relevance of these proteins, there is a lack of detailed structural information about both the large complexes formed in the native state and in the cataract- related aggregates. Determining these structures will require new biophysical and analytical methods. The objective of this proposal is to clarify the molecular basis of cataract formation. The specific molecular target is ?S-crystallin, a major structural component of the eye lens. New methodology will be developed in order to investigate the structural factors related to ?S-crystallin stability and solubility, primarily in solid-state NMR. Phage display will be used to identify peptides that specifically bind to misfolded crystallin variants and to discover new aggregation-prone variants themselves. These peptide binders will be used in preliminary NMR experiments along the way to full structure determination. Differential isotope labeling of peptide binders and variant crystallins can be used to identify crystallin residues involved in altered intermolecular interactions and provide preliminary structural information. A novel high-field 1H,13C,2H,15N solid- state NMR probe will be designed and built in order to perform 2H-detected experiments currently not possible with available probes. New experiments taking advantage of this unique instrumentation will be developed to investigate crystallin aggregates and other solid but highly mobile samples. We will also make use of recent advances in solid-state NMR to determine the molecular structures of wild-type ?S-crystallin at high concentration, aggregates of the G18V variant associated with congenital cataracts in humans, and potentially other variants found using phage display. The G18V variant has been demonstrated to be biologically relevant and therefore will serve as a starting point for our investigations into structure/function relationships in the healthy and cataract states of eye lens proteins. Elucidation of these structures will improve our understanding of how cataracts form and may lead to novel strategies for their prevention and treatment. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to determine how the structural eye lens protein ?S- crystallin aggregates, leading to cataract formation. We will develop biochemical methods to identify misfolded protein variants, and develop new solid-state NMR instrumentation and techniques to determine the molecular structures of both the healthy and cataract states of ?S-crystallin. Elucidation of these structures will improve our understanding of how cataracts form and may lead to novel strategies for their prevention and treatment.