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. H-1 NMR spectroscopy has shown that mammalian sHsps possess short , polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some d ifferences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscop y 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 alpha-lactalbumin against precipitation following reduction w ith dithiothreitol. The accessible hydrophobic surface of the mutant protei n is less than that of the wild-type protein and the mutant is also less st able at elevated temperature. H-1 NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C-terminal f lexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced alpha-lactalbumin by H-1 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.