Mouse Hsp25, a small heat shock protein. The role of its C-terminal extension in oligomerization and chaperone action

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. ¹H NMR spectroscopy has shown that mammalian sHsps possess short, polar anti highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild- type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize α-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. ¹H NMR spectroscopy reveals that deletion of the C- terminal extension of Hsp25 leads to induction of extra C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced α-lactalbumin by ¹H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity.