Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity.
During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.
Gustavsson
N
N
Department of Biochemistry, Lund University, S-221 00 Lund, Sweden.
Kokke
B P
BP
Anzelius
B
B
Boelens
W C
WC
Sundby
C
C
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins; D002736:Chloroplasts / Q000378:metabolism*; D002942:Circular Dichroism; D017124:Conserved Sequence; D005453:Fluorescence; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008027:Light; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013696:Temperature; D013816:Thermodynamics
D029681:Arabidopsis Proteins; C400143:HSP21 protein, Arabidopsis; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010940:Plant Proteins; D008715:Methionine; D007930:Leucine