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

Using genetic code expansion (GCE), we incorporated an oxidation 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 and experiments suggests a potential mechanism for polymerization as well as oxidation-induced conformational changes that may cause susceptibility to thermal aggregation. Our findings highlight the utility of GCE platforms for systematically evaluating the impact of post-translational modifications on disease-related proteins. All data collected and mentioned either in the main text or SI have been uploaded here.

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