Effects of site-directed mutations on the chaperone-like activity of alpha B-crystallin

Recombinant alpha B-crystallin has been shown to exhibit chaperone-like activity, suppressing the thermal aggregation of gamma-crystallin and aggregation of the reduced insulin B chain conferring thermotolerance to Escherichia coli BL21(DE3) cells. Mutations were made in three specific areas of the alpha B-crystallin, the N terminus D2G, the conserved phenylalanine-rich region, F24R, F27R, F27A, and the two C-terminal lysines K174L/K175L, K174G/K175G. Biophysical characterization of the mutant alpha B-crystallins using far-UV CD revealed no change in secondary structural elements. Tryptophan fluorescence demonstrated global structural changes. Heat stability of the mutant alpha B-crystallins was not significantly affected as indicated by tryptophan fluorescence of heat-treated proteins. Mutations within the phenylalanine-rich region abolish the chaperone-like activity as measured by bath in vivo and in vitro assays. Proteins with mutations at the C terminus demonstrated no significant chaperone-like activity, failing to confer thermotolerance on E. coli and demonstrating no significant inhibition of protein aggregation in either gamma-crystallin or reduced insulin B chain assays. The N-terminal mutation D2G demonstrated a significant reduction in efficiency of the chaperone-like activity although some thermotolerance was conferred in the E. coli assay. In vitro assays showed that complete inhibition of aggregation was only achieved at 10-fold higher concentrations of D2G than that required by the native alpha B-crystallin. Consistent changes in the chaperone-like activity of the site-directed mutants were demonstrated by the three assays. The results suggested that both charge-charge and hydrophobic interactions are important in protein binding by alpha B-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.