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Investigation of gamma-crystallin biophysical properties and aggregation pathways relevant to cataract formation

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This thesis examines the biophysical properties of beta/gamma-crystallin proteins with a specific focus on how exogenous factors such as UV and divalent cations drive the aggregation of eye lens gammaS-crystallin. Beta/gamma-crystallin superfamily proteins are found in archaeal, bacterial, and eukaryotic species, with varying functionalities that represent evolutionary adaptation. In this work the eye lens gammaS-crystallin is shown to have orthogonal behavior to Ciona intestinalis beta/gamma-crystallin (Ci-b/g) – a calcium binding protein – in the presence of various divalent cations. Ci-b/g coordinates divalent cations at the same site, increasing its thermal stability, whereas cystine coordination to some transition metal divalent cations induces aggregation in gammaS-crystallin. Unlike other transitional metals, copper induced aggregation of gammaS is buffered by the presence of free, solvent accessible cysteines. Copper-driven aggregation differs from other underlying causes such as UV or missense point mutations and may be attributed to a combination of different factors. Notably, copper is able to catalyze intermolecular disulfide bond formation as well as facilitate radical crosslinking. Reductions in beta-sheet content also occur in aggregated species, further suggesting that hydrophobic interactions may drive aggregation. Another physiologically relevant underlying mechanism, UV radiation, was used to induced aggregates for comparative measurements with acid-induced fibrils that exhibit amyloid character. The two groups are distinct in their biophysical characterization. High levels of turbidity but low thioflavin T binding for UV-induced aggregates while acid-induced fibrils exhibit the opposite characteristics. In both cases, it is notable that mutations compromising stability exacerbate the observable characterization. These results suggest that gamma-crystallin aggregates resulting from accumulated UV exposure are unlikely to contain fibrillar character.

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