The human eye lens comprises transparent fiber cells whose nuclei and organelles are lost during development, resulting in a protein matrix mainly made of crystallins. These proteins are long-lived and must remain soluble to maintain lens transparency. Over time, crystallins undergo different post-translational modifications that can reduce solubility and cause aggregation, as lens cells cannot regenerate or degrade proteins. It leads to cataract formation, a leading cause of blindness. Oxidative stress, specifically from UV radiation, plays a major role in age-related cataracts by modifying amino acid residues, including tryptophan, which is important for the stability of crystallin. In γS-crystallin, tryptophan at position 163 is particularly susceptible to oxidation, which makes it important to study its impact on protein structure and aggregation to better understand cataract development.
This study aimed to assess how site-specific oxidation of the W163 residue in γS-crystallin affects the protein’s structure, stability, and aggregation behavior. The researchers used hydroxytryptophan (5HTP) as a model oxidative modification to mimic the oxidation of tryptophan and evaluate its impact on the physicochemical properties of the protein.
The researchers introduced 5HTP at position 163 of γS-crystallin using genetic code expansion technology. An amber stop codon was inserted in the gene at the W163 position, which allows incorporation of the unnatural amino acid 5HTP during protein expression in engineered Escherichia coli cells. Both the wild-type protein (γS-WT) and the modified variant (γS-W163(5HTP)) were expressed and purified using nickel affinity chromatography followed by size-exclusion chromatography. Successful incorporation of the modified residue was confirmed using liquid chromatography-mass spectrometry.
Structural characteristics of the proteins were analyzed by circular dichroism spectroscopy to assess secondary structure and intrinsic tryptophan fluorescence to evaluate the environment of the hydrophobic core. Chemical denaturation experiments using guanidine hydrochloride and thermal unfolding experiments were performed to measure protein stability. Dynamic light scattering was used to monitor aggregation behavior by measuring changes in particle size with increasing temperature. Molecular dynamics simulations were conducted to model protein interactions at different temperatures and explore possible mechanisms of aggregation. Solution-state nuclear magnetic resonance spectroscopy was used to examine residue-level structural changes and alterations in hydrogen-bonding interactions caused by the oxidation mimic.
Structural analysis showed that incorporation of 5HTP at position 163 produced only minimal changes to the overall structure of γS-crystallin at room temperature. Circular dichroism spectra of the modified protein closely resembled those of the wild-type protein, which indicates that the characteristic β-sheet structure of γ-crystallins remained largely intact. Intrinsic fluorescence measurements showed only a slight shift in emission wavelength and a minor narrowing of the emission peak, which suggests a slight change in the local environment around the modified residue but no major structural disruption.
Despite these minor structural changes, the oxidized variant showed significantly reduced stability. Chemical unfolding experiments demonstrated that γS-W163(5HTP) unfolded at lower concentrations of guanidine hydrochloride than the wild-type protein. Thermal unfolding studies revealed that the melting temperature decreased from approximately 78.1°C in the wild-type protein to 72.8°C in the modified variant, demonstrating that oxidation of W163 weakens the structural stability of γS-crystallin.
Dynamic light scattering experiments indicated that the modified protein aggregated at lower temperatures (34°C) compared with the wild-type protein (43°C). Molecular dynamics simulations suggested that although both proteins exist as dimers at physiological temperatures, the wild type of protein dissociates into monomers at higher temperatures, whereas the modified variant forms larger oligomers. Nuclear magnetic resonance spectroscopy revealed minor chemical shift changes, confirming that the global structure remained intact but indicated localized perturbations near R158 and A166, which suggest increased flexibility and potential transient unfolding.
Overall, the study shows that oxidation of a single tryptophan residue in γS-crystallin can influence protein stability and aggregation without dramatically altering its overall structure. Incorporation of 5-hydroxytryptophan at position 163 produced only minor structural changes under normal conditions but reduced thermal and chemical stability and significantly increased the tendency of the protein to aggregate. Molecular simulations and NMR analysis suggest that this effect occurs from subtle local structural rearrangements that modify intermolecular interactions and create aggregation-prone surfaces. These findings highlight the important role of tryptophan residues in maintaining the stability of crystallin proteins and provide insights into how oxidative modification accumulated over time may contribute to protein aggregation in the eye lens and development of cataracts.
Reference: Seo Y, Long ZG, Demerdjian TK, Avenido AA, Butts CT, Martin RW. Mimicking oxidative damage in γS-crystallin with site-specific incorporation of 5-hydroxytryptophan. Biophys Rep (N Y). 2026. doi:10.1016/j.bpr.2026.100251
