Evidence for an essential function of the N terminus of a small heat shock protein in vivo , independent of in vitro chaperone activity

To investigate the mechanism of small heat shock protein (sHsp) function, unbiased by current models of sHsp chaperone activity, we performed a screen for mutations of Synechocystis Hsp16.6 that reduced the ability of the protein to provide thermotolerance in vivo . Missense mutations at 17 positions throughout the protein and a C-terminal truncation of 5 aa were identified, representing the largest collection of sHsp mutants impaired in function in vivo . Ten mutant proteins were purified and tested for alterations in native oligomeric structure and in vitro chaperone activity. These biochemical assays separated the mutants into two groups. The C-terminal truncation and six mutations in the α-crystallin domain destabilized the sHsp oligomer and reduced in vitro chaperone activity. In contrast, the other three mutations had little effect on oligomer stability or chaperone activity in vitro . These mutations were clustered in the N terminus of Hsp16.6, pointing to a previously unrecognized, important function for this evolutionarily variable domain. Furthermore, the fact that the N-terminal mutations were impaired in function in vivo , but active as chaperones in vitro , indicates that current biochemical assays do not adequately measure essential features of the sHsp mechanism of action.