The structural stability and chaperone activity of artemin, a ferritin homologue from diapause-destined Artemia embryos, depend on different cysteine residues.
Diapause-destined embryos of the crustacean, Artemia franciscana, accumulate large amounts of an oligomeric, heat-stable, molecular chaperone termed artemin, a cysteine-enriched ferritin homologue. In this study, cysteines 22, 61, 166, and 172 of artemin were substituted with alanines, respectively yielding ArtC22A, ArtC61A, ArtC166A, and ArtC172A. Wild-type and modified artemins were synthesized in transformed bacteria and purified. As measured by heat-induced denaturation of citrate synthase in vitro, each substitution reduced chaperone activity, with ArtC172A the least active. Protein modeling indicated that C172 is close to a region of surface hydrophobicity, also present in ferritin, suggesting that this site contributes to chaperone activity. Only slight differences in oligomer molecular mass were apparent between artemin variants, but ArtC22A and ArtC61A displayed significantly reduced thermostability, perhaps due to the disruption of an inter-subunit disulphide bridge. In contrast, ArtC172A was thermostable, reflecting the location of C172 on the oligomer surface and that it contributes minimally to artemin stabilization. To our knowledge, this is the initial study of structure/function relationships within a ferritin homologue of importance in diapause and the first to indicate that a defined region of hydrophobicity contributes to artemin and ferritin chaperoning.
Hu
Yan
Y
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
Bojikova-Fournier
Svetla
S
King
Allison M
AM
MacRae
Thomas H
TH
D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia; D060829:Arthropod Proteins; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004625:Embryo, Nonmammalian; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D007447:Invertebrate Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033862:Iron-Binding Proteins; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics; D055550:Protein Stability; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D033862:Iron-Binding Proteins; D018832:Molecular Chaperones; D009479:Neuropeptides; D016601:RNA-Binding Proteins; C490888:artemin protein, brine shrimp; D005293:Ferritins; C075027:diapause hormone; D003545:Cysteine