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Site-specific modifications of aspartate residues spontaneously occur in crystallin, the major protein in the lens. One of the primary modification sites is Asp151 in αA-crystallin. Isomerization and racemization alter the crystallin backbone structure, reducing its stability by inducing abnormal crystallin–crystallin interactions and ultimately leading to the insolubilization of crystallin complexes. These changes are considered significant factors in the formation of senile cataracts. However, the mechanisms driving spontaneous isomerization and racemization have not been experimentally demonstrated. In this study, we generated αA-crystallins with different homo-oligomeric sizes and/or containing an asparagine residue at position 151, which is more prone to isomerization and racemization. We characterized their structure, hydrophobicity, chaperone-like function, and heat stability, and examined their propensity for isomerization and racemization. The results show that the two differently sized αA-crystallin variants possessed similar secondary structures but exhibited different chaperone-like functions depending on their oligomeric sizes. The rate of isomerization and racemization of Asp151, as assessed by the deamidation of Asn151, was also found to depend on the oligomeric sizes of αA-crystallin. The predominant isomerization product via deamidation of Asn151 in the different-sized αA-crystallin variants was L-β-Asp in vitro, while various modifications occurred around Asp151 in vivo. The disparity between the findings of this in vitro study and in vivo studies suggests that the isomerization of Asp151 in vivo may be more complex than what occurs in vitro. [ABSTRACT FROM AUTHOR]

Conserved tryptophan residues are critical for the structure and the stability of β/γ‐crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in β/γ‐crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of β/γ‐crystallin, which correlates with age‐related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human βB2‐crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo‐oligomerization of βB2‐crystallin—W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of βB2‐crystallin vary over time. By using SAXS, we found that the dimer of βB2‐crystallin in solution resembled the lattice βB1‐crystallin dimer (face‐en‐face), whereas the tetramer of βB2‐crystallin in solution resembled its lattice tetramer (domain‐swapped). Our results suggest that homo‐oligomerization of βB2‐crystallin includes potential inter‐subunit reactions, such as dissociation, unfolding, and re‐formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of βB2‐crystallin in lens. [ABSTRACT FROM AUTHOR]