title: Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin.
abstract: Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.
authors.affiliation: Department of Biology, Ashland University, Ashland, Ohio, United States of America. mposner@ashland.edu
authors.forename: Mason
authors.initials: M
authors.lastname: Posner
authors.forename: Andor J
authors.initials: AJ
authors.lastname: Kiss
authors.forename: Jackie
authors.initials: J
authors.lastname: Skiba
authors.forename: Amy
authors.initials: A
authors.lastname: Drossman
authors.forename: Monika B
authors.initials: MB
authors.lastname: Dolinska
authors.forename: J Fielding
authors.initials: JF
authors.lastname: Hejtmancik
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Sergeev
mesh_terms: D000064:Acclimatization; D000595:Amino Acid Sequence; D000818:Animals; D005399:Fishes; D050888:Heat-Shock Proteins, Small / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050888:Heat-Shock Proteins, Small; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain