Abstract The eye lens crystallins, which maintain the transparency of the eye lens by providing a well-defined gradient of refractive index, are a scientifically important and medically relevant group of proteins. In contrast to most other proteins, which are constantly subject to degradation and recycling, crystallins have very low turnover and must remain intact for a lifetime. This is even more remarkable considering their extremely high concentration in the lens. Cataract, a major cause of blindness worldwide, results when the structural crystallins aggregate or phase-separate, rendering the lens opaque. Over time, protein degradation 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. Furthermore, several known point mutations cause hereditary juvenile-onset cataracts. Because of the medical and biophysical relevance of crystallins, there is a need for detailed structural information about both the large complexes formed in the native state and in the cataract- related aggregates. Molecular-level characterization of crystallin aggregation at the level of detail required to guide the design of new therapeutic strategies requires the development of instrumentation and methodology. The objective of this project is to clarify the molecular basis of the crystallin aggregation that leads to cataract formation. The major types of crystallins can be categorized as either structural (b/g) or solubilizing (a). The specific molecular target is gS-crystallin, a major structural component of the eye lens, and its interactions with the α-crystallin chaperones. New NMR methodology will be developed to investigate the structural factors related to gS-crystallin stability and solubility, primarily in the solid state. 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. We have designed and built a novel high-field 1H,13C,2H,15N solid-state NMR probe to perform 2H-detected experiments not possible with previously available probes. Building on this success, new experiments that make use of this unique instrumentation will be developed to investigate crystallin aggregates and other solid but highly mobile samples. We will continue to utilize recent advances in solid-state NMR to investigate molecular structure and dynamics in wild-type gS- crystallin at high concentration, aggregates of variants associated with congenital cataracts in humans, as well as aggregates formed by UV irradiation and binding of metal cations. The G18V variant serves as a starting point for our investigations into structure/function relationships in the healthy and cataract states of eye lens proteins; however, in the later stages of the project, the focus of the work will shift to models for age-related cataract, which affects many more patients. Elucidation of these structures will improve our understanding of how cataract formation and guide the development of novel strategies for their prevention and treatment.

Project Narrative The objective of this proposal is to determine how the structural eye lens protein γS- crystallin aggregates, leading to cataract formation. We will identify aggregation-prone protein variants, characterize the types of aggregates formed, and develop solid-state NMR instrumentation and techniques to determine the molecular structures of both the healthy and cataract states of gS-crystallin. Elucidation of these structures will improve our understanding of cataract formation, potentially leading to novel strategies for their prevention and treatment.