Mini-αA-crystallin protects a client lens protein from catastrophic aggregation due to heat stress.

Abstract

The clarity and refractivity of the eye lens are mediated by the highly soluble crystallin proteins. Post-translational modifications impact solubility and stability of the structural and refractive βγ-crystallins, eventually leading to cataract. Such damaged proteins are kept in solution by the holdase chaperone α-crystallins, maintaining lens transparency over decades despite the absence of protein turnover. It was previously found that a short peptide from human αA-crystallin (mini-αA-crystallin [MAAC]) retains some chaperone activity, with hydrophobic interactions hypothesized to mediate chaperone-client interactions; MAAC has been hypothesized to have β-strand structure in solution, although its conformational ensemble under these conditions has not been well-characterized. Here, we employ a combination of nuclear magnetic resonance (NMR), circular dichroism spectroscopy, dynamic light scattering, and molecular dynamics simulations to examine the behavior of MAAC in dilute solution and in combination with human γS-crystallin. Structural ensembles of two alanine variants of MAAC (I4A and L6A) show that the variants lack well-defined secondary structure, but have a preference for a bent conformation with some self-interaction. A partial alanine scan indicates that several hydrophobic residues are important for peptide solubility, also modifying the peptide's conformational ensemble. Tests of wild-type MAAC chaperone activity on thermally stressed γS-crystallin show little interaction between MAAC and the client protein below its unfolding temperature. However, MAAC does inhibit large-scale aggregation at the γS-crystallin unfolding temperature. NMR measurements indicate only weak, transient interaction with the client protein during the intermediate aggregation phase, suggesting a sharp phase transition in the MAAC-client system.