BPS2025 - Q147E deamidation in αA-crystallin increased membrane binding whereas cholesterol inhibited such binding

α-Crystallin (αABc) is the primary molecular chaperone found in the eye lens and is an essential component in maintaining the solubility of the eye lens proteins. However, with age and cataract formation, αABc membrane binding increased, and the αABc subunits, αA-crystallin (αAc) and αB-crystallin (αBc), undergo various post-translational modifications (PTMs), with the most prevalent PTMs being deamidation. It has been reported that Q147E deamidation in αAc (Q147E-αAc) is significantly higher in cataractous lenses compared to age-matched healthy lenses, suggesting the causal link between Q147E-αAc and cataract development. However, the role of Q147E-αAc in the membrane binding remains unclear. This research aims to elucidate the effects of Q147E-αAc on membrane binding and the role of cholesterol (Chol) in modulating such binding. Experimentation involved preparing Chol/1-palmitoyl-2-oleoylphosphatidylcholine (Chol/POPC) membranes containing 2 mol% cholesterol analog spin label (CSL), which incorporates into the membranes near the lipid headgroup region and using the electron paramagnetic resonance spin-labeling method to probe αAc binding to the membrane. Our results indicate Q147E-αAc promotes a higher maximum parentage membrane surface occupied by Q147E-αAc to our investigated membranes than that seen for wild-type αAc (WT-αAc). Adding Chol at a Chol/POPC mixing ratio of 0.5 reduces the binding of WT-αAc and Q147E-αAc and completely inhibits the membrane binding at a mixing ratio of 1.5. WT-αAc and Q147E-αAc membrane binding decreases mobility while slightly increasing membrane order and a significant increase in hydrophobicity near the headgroup region, creating a hydrophobicity barrier for the passage of polar molecules. However, adding Chol diminishes the hydrophobicity increase seen with binding, suggesting Chol inhibits the binding of WT-αAc and Q147E-αA, preventing the formation of a hydrophobic barrier and potentially protecting against cataract formation.