Tryptophan oxidation-induced dynamics highlight why stable γS-crystallin variants aggregate.

Human γS-crystallin (γS) is a highly soluble protein found in the eye lens, where it is densely packed with other proteins to form the refractive tissue required for vision. Mutations in γS can compromise its solubility and cause congenital cataract, while post-translational modifications (PTMs) can lead to age-related cataract. γS is especially susceptible to oxidative PTMs, which accumulate from lifelong exposure to UV light. We previously investigated the effects of side chain oxidation at tryptophan 163 (W163) by incorporating an oxidation mimic, 5-hydroxytryptophan (5HTP), in a site-specific manner using genetic code expansion. The resulting variant, γS-W163(5HTP), retains the overall fold of the wild-type γS (γS-WT) even under thermal and chemical stress, yet readily forms large oligomers at physiological temperature. Here, we probe the mechanism underlying this behavior. We first characterize γS-W163S, a serine variant in which the indole ring at position 163 is effectively removed, to further define the role of W163 in γS-crystallin structure and stability. We also map changes in backbone dynamics caused by W163 oxidation in γS-W163(5HTP) using solution-state NMR. Together, our findings support the established model in which crystallin aggregation is decoupled from global destabilization: although the oxidative modification at W163 is structurally tolerated through rearrangement of the local hydrogen bond network, the resulting changes in backbone flexibility render aggregation-competent conformations accessible. This work provides mechanistic insights into how covalent damage to crystallin monomers promotes aberrant intermolecular interactions that lead to cataract formation.