title: Molecular chaperone function of the Rana catesbeiana small heat shock protein, hsp30.
abstract: Eukaryotic small heat shock proteins (shps) act as molecular chaperones by binding to denaturing proteins, preventing their heat-induced aggregation and maintaining their solubility until they can be refolded back to their normal state by other chaperones. In this study we report on the functional characterization of a developmentally regulated shsp, hsp30, from the American bullfrog, Rana catesbeiana. An expression vector containing the open reading frame of the hsp30 gene was expressed in Escherichia coli. Purified recombinant hsp30 was recovered as multimeric complexes and was composed of a mixture of alpha-helical and beta-sheet-like structures as determined by circular dichroism analysis. Hsp30 displayed chaperone activity since it inhibited heat-induced aggregation of citrate synthase. Furthermore hsp30 maintained heat-treated luciferase in a folding competent state. For example, heat denatured luciferase when microinjected into Xenopus oocytes did not regain enzyme activity whereas luciferase heat denatured with hsp30 regained 100% enzyme activity. Finally, hsp30 protected the DNA restriction endonuclease, PstI, from heat inactivation. PstI incubated alone at 42 degrees C lost its enzymatic function after 1 h whereas PstI supplemented with hsp30 accurately digested plasmid DNA after 4 h at the elevated temperature. These results clearly indicate a molecular chaperone role for R. catesbeiana hsp30.
authors.affiliation: Department of Biology, University of Waterloo, Ontario, Canada, N2L 3G1.
authors.forename: Angelo
authors.initials: A
authors.lastname: Kaldis
authors.forename: Burr G
authors.initials: BG
authors.lastname: Atkinson
authors.forename: John J
authors.initials: JJ
authors.lastname: Heikkila
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D002950:Citrate (si)-Synthase / Q000737:chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D006358:Hot Temperature; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014981:Xenopus
chemical_list: D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D004247:DNA; D008156:Luciferases; D002950:Citrate (si)-Synthase; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific