Effects of site-directed mutations on the chaperone-like activity of alphaB-crystallin.
Recombinant alphaB-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 alphaB-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 alphaB-crystallins using far-UV CD revealed no change in secondary structural elements. Tryptophan fluorescence demonstrated global structural changes. Heat stability of the mutant alphaB-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 both 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 alphaB-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 alphaB-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.
Wolfson Laboratory, School of Animal and Microbial Sciences, The University of Reading, Whiteknights, Reading, Berkshire RG6 6AJ, United Kingdom.
M L
ML
Plater
D
D
Goode
M J
MJ
Crabbe
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D003459:Crystallins / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000235:genetics*; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones