Site-directed mutations within the core "α-crystallin" domain of the small heat-shock protein, human αB-crystallin, decrease molecular chaperone functions

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core "alpha-crystallin" domain of the small heat-shock protein (sHsp) and molecular chaperone, human alpha B-crystallin. Five alpha B-crystallin mutants containing single amino acid substitutions within the core a-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E, coli at 50 degrees C in vivo. Ln contrast, seven aB-crystallin mutants containing substitutions N-terminal to the core a-crystallin domain generally resembled wild-type alpha B-crystallin in chaperone activity in vitro and in vivo. Size-exclusion chromatography, ultraviolet circular dichroism spectroscopy and limited proteolysis were used to evaluate potential structural changes in the 12 alpha B-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core alpha-crystallin domain were similar to wild-type alpha B-crystallin. SDS-PAGE patterns of chymotryptic digestion were also similar in the mutant and wild-type proteins, indicating that the mutations did not introduce structural modifications that altered the exposure of proteolytic cleavage sites in alpha B-crystallin. On the basis of the similarities between the sequences of human alpha B-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alpha B-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alpha B-crystallin identify regions that may be important for the molecular chaperone functions of sHsps. (C) 1999 Academic Press.