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Volume 271, Number 45, Issue of November 8, 1996 pp. 28558-28566
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Effects of Site-directed Mutations on the Chaperone-like Activity of B-Crystallin

(Received for publication, May 30, 1996, and in revised form, August 12, 1996)

From the Wolfson Laboratory, School of Animal and Microbial Sciences, The University of Reading, Whiteknights, Reading, Berkshire RG6 6AJ, United Kingdom

Recombinant B-crystallin has been shown to exhibit chaperone-like activity, suppressing the thermal aggregation of -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 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 B-crystallins using far-UV CD revealed no change in secondary structural elements. Tryptophan fluorescence demonstrated global structural changes. Heat stability of the mutant 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 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 -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 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 B-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.

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