Immobilization of the C-terminal extension of bovine αA-crystallin reduces chaperone-like activity

Abstract

α-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of α-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. α- Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that α-crystallin subunits have a polar and unstructured C- terminal extension that functions as a sort of solubilizer. In this paper we have described five αA-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of αA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.