IMMOBILIZATION OF THE C-TERMINAL EXTENSION OF BOVINE ALPHA-A-CRYSTALLIN REDUCES CHAPERONE-LIKE ACTIVITY

alpha-Crystallins occur as multimeric complexes, which are able to sup press precipitation of unfolding proteins. Although the mechanism of t his chaperone-like activity is unknown, the affinity of alpha-crystall in for aggregation-prone proteins is probably based on hydrophobic int eractions. alpha-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 alpha-crystallin subunit s have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five alpha A-c rystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspart ate does not substantially influence chaperone-like activity. In contr ast, introduction of a hydrophobic tryptophan greatly diminishes funct ional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the insert ed tryptophan is located in a polar environment. However, NMR spectros copy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furt hermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing t he polarity of the C-terminal extension of alpha A-crystallin by inser tion of a highly hydrophobic residue can seriously disturb structural and functional integrity.