The human eye lens plays an essential role in vision by focusing light onto the retina. This transparent tissue consists of densely packed crystallin proteins that exhibit remarkable solubility despite minimal protein turnover. Post-translational damages that accrue over a lifetime can reduce crystallin solubility, resulting in the precipitation or phase-separation of protein aggregates. Oxidation is a common type of modification that can cause such opacification of the lens, particularly in age-related cataracts. This type of damage is difficult to study using biophysical methods, as it is often randomly dispersed throughout the protein. Here, we used genetic code expansion (GCE) to study the oxidation of W163 in human γS-crystallin, a structural lens protein particularly susceptible to oxidation. Building on our previous finding that this residue becomes oxidized upon γ-irradiation, we incorporated a model oxidation product, 5-hydroxytryptophan (5-HTP), at residue 163 of γS-crystallin using a GCE platform. The resulting oxidized variant was characterized using optical spectroscopy, mass spectrometry and NMR. Compared to wild-type, the oxidized variant showed diminished resilience against thermal and chemical stress, although it had similar properties under ambient conditions. Notably, its aggregation was triggered at close to physiological temperature, compared to much higher thermal resistance in wild-type. The underlying conformational changes and possible aggregation modes were investigated using molecular dynamics simulations and NMR spectroscopy. Our findings highlight the utility of GCE platforms for systematically evaluating site-specific post-translational modifications and provide insight into the molecular mechanisms of age-related cataracts.
Seo et al. (Sun,) studied this question.