Abstract: 

The human eye lens maintains a high refractive index despite extremely high protein concentrations and minimal protein turnover; this requires crystallin proteins to remain soluble over a lifetime. Precipitation or phase separation of crystallins leads to cataract, a leading cause of blindness if untreated. Age-related cataract in particular is strongly linked to accumulated post-translational modifications, especially oxidative damage arising from lifelong UV exposure. γS-crystallin is a highly soluble βγ-crystallin family member that contributes to lens transparency but is vulnerable to oxidative stress. Oxidation at tryptophan 163 (W163) has been detected in irradiated γS samples and cataractous lenses, although its mechanistic impact on structure and aggregation remained unclear. Here, I investigate how site-specific oxidation at W163 influences γS-crystallin stability and solubility, leveraging genetic code expansion to incorporate 5-hydroxytryptophan (5HTP) as an oxidation mimic. I show that the oxidized variant (γS-W163(5HTP)) remains globally folded yet exhibits reduced stability and undergoes premature oligomerization below physiological temperatures, whereas the wild-type γS (γS-WT) remains largely monomeric under comparable conditions. Using biophysical characterization, including NMR spectroscopy, I conclude that aggregation can be initiated without global unfolding, as oxidation at W163 instead perturbs the local hydrogen-bonding network and redistributes backbone dynamics in a manner that increases transient unfolding events and enables intermolecular association. To further probe the role of this site, a W163S variant (γS-W163S) was characterized as a structural analogue that removes the aromatic indole ring. Although this mutant is significantly destabilized, it does not exhibit the same premature aggregation behavior, indicating that stability and aggregation propensity are decoupled in this system. Overall, these findings show that site-specific oxidative modifications can tune protein dynamics to populate aggregation-prone states, thereby refining models of crystallin aggregation in cataract formation.