title: Structure-based analysis of the beta8 interactive sequence of human alphaB crystallin.
abstract: The functional importance of the beta8 sequence ((131)LTITSSLS(138)), which is on the surface of the alpha crystallin core domain of human alphaB crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (alphaAbeta8 and CEbeta8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the beta8 mutants to be 6-8 subunits larger than wt alphaB crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the beta8 sequence was similar to wt alphaB crystallin when beta(L) crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CEbeta8 mutants. The prominent location of Thr-134, Ser-136, and Ser-138 on the exposed surface of the alpha crystallin core domain could account for the effect on complex assembly and chaperone activity. Modulation of chaperone activity by the exposed residues of the beta8 sequence in the alpha crystallin core domain was independent of complex size. The results established the beta3-beta8-beta9 surface of the alpha crystallin core domain as an interface for complex assembly and chaperone activity.
authors.affiliation: Biomolecular Structure and Design, University of Washington, Seattle, Washington 98195-7420, USA.
authors.forename: Joy G
authors.initials: JG
authors.lastname: Ghosh
authors.forename: Marcus R
authors.initials: MR
authors.lastname: Estrada
authors.forename: John I
authors.initials: JI
authors.lastname: Clark
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D038203:alpha-Crystallin B Chain / Q000737:chemistry*
chemical_list: D038203:alpha-Crystallin B Chain