Abstract
We previously demonstrated that deletion of the (54)FLRAPSW(61) sequence, containing the key phosphorylation site serine 59 (S59), resulted in a two-fold reduction in oligomeric mass and a ten-fold enhancement of αB-crystallin's chaperone activity. This study examined whether targeted deletion (ΔS59) or phosphomimetic substitution (S59D) could replicate these effects. Using MALS analysis, we found that the average oligomeric mass decreased from 579 kDa in the wild type (αB-WT) to 556 kDa in αB-ΔS59 and 434 kDa in αB-S59D. Interestingly, the αB-S59A variant had an increased mass of 611 kDa. All variants retained their chaperone function, but their efficiencies varied significantly. Specifically, αB-S59D formed smaller, more polydisperse complexes that effectively suppressed aggregation when interacting with rapidly aggregating substrates. In contrast, αB-ΔS59 and αB-S59A created stable complexes with lysozyme, reducing precipitation and aggregate size. Zeta potential measurements revealed distinct surface charge profiles among the variants; however, no clear correlation was observed between these charges and their chaperone efficiency. Additionally, cytotoxicity assays conducted on ARPE-19 cells under oxidative stress showed that all S59 variants exhibited comparable protective effects against cell death relative to αB-WT. These results indicate that while S59 is not essential for oligomer formation or chaperone function, it plays a crucial role in modulating oligomer size and interactions with various substrates. Notably, the effects of αB-S59D were measurable but did not replicate the enhanced functionality observed with the complete deletion of the 54-61 motif, reinforcing the significance of the N-terminal region.