Mimicking oxidative damage in γS-crystallin with site-specific incorporation of 5-hydroxytryptophan

ABSTRACT

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 solu- bility despite minimal protein turnover. Post-translational modifications that accumulate over a lifetime can reduce crystallin solubility, resulting in the precipitation or phase-separation of pro- tein aggregates. Oxidation is a common type of modification that can cause such opacification of the lens, particularly in age-related cataract. Here, we study the oxidation of W163 in γS- crystallin, a structural lens protein that is particularly vulnerable to oxidative stress. We were motivated by previous findings, which report the oxidation of this residue in diseased as well as UV- and γ-irradiated samples. Using genetic code expansion (GCE), we incorporated an oxi- dation mimic, 5-hydroxytryptophan (5HTP), at position 163 of γS-crystallin (γS-W163(5HTP)). This subtle change in the structural and electronic properties of its side-chain is hypothesized to destabilize the hydrophobic core of the C-terminal domain. γS-W163(5HTP) was characterized and compared to the wild-type (γS-WT). Although the overall fold and stability of the two proteins were comparable, the aggregation of γS-W163(5HTP) was triggered at notably lower temperatures compared to γS-WT. Subsequent investigation of this observation using both simulations andexperiments suggests a potential mechanism for polymerization as well as oxidation-induced conformational changes that may cause susceptibility to thermal aggregation. Our findings high- light the utility of GCE platforms for systematically evaluating the impact of post-translational modifications on disease-related proteins.