title: Site-directed mutations within the core "alpha-crystallin" domain of the small heat-shock protein, human alphaB-crystallin, decrease molecular chaperone functions.
abstract: 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 alphaB-crystallin. Five alphaB-crystallin mutants containing single amino acid substitutions within the core alpha-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. In contrast, seven alphaB-crystallin mutants containing substitutions N-terminal to the core alpha-crystallin domain generally resembled wild-type alphaB-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 alphaB-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 alphaB-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 alphaB-crystallin. On the basis of the similarities between the sequences of human alphaB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alphaB-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alphaB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps.
authors.affiliation: Department of Biological Structure, University of Washington, Seattle, WA, 98195-7420, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Muchowski
authors.forename: G J
authors.initials: GJ
authors.lastname: Wu
authors.forename: J J
authors.initials: JJ
authors.lastname: Liang
authors.forename: E T
authors.initials: ET
authors.lastname: Adman
authors.forename: J I
authors.initials: JI
authors.lastname: Clark
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D017017:Methanococcus / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D019843:Archaeal Proteins; D003459:Crystallins; C113795:HSP16.5 protein, Methanococcus jannaschii; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins