부산대학교 도서관

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core “α-crystallin” domain of the small heat-shock protein (sHsp) and molecular chaperone, human αB-crystallin. Five αB-crystallin mutants containing single amino acid substitutions within the core α-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E. coli at 50 °C in vivo. In contrast, seven αB-crystallin mutants containing substitutions N-terminal to the core α-crystallin domain generally resembled wild-type αB-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 αB-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core α-crystallin domain were similar to wild-type αB-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 αB-crystallin. On the basis of the similarities between the sequences of human αB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for αB-crystallin was constructed. The mutations at sites within the core α-crystallin domain of αB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps.