A Chloroplast Localized Small Heat Shock Protein, Hsp21. Importance of Hsp21 in stress protection of transgenic Arabidopsis thaliana, recombinant expression and characterization of oxidation-dependent conformational changes

Abstract: A specific group of heat shock proteins, the so called small heat shock proteins (sHsps), exists in all organisms but are especially abundant in plants. The sHsps prevent aggregation of other proteins (substrate proteins) during transient heat and oxidative stress. Molten globule forms of the substrate proteins are kept in a re-folding competent state by binding onto the outer surface of the sHsp. Members of the superfamily of sHsps and a-crystallins share a conserved C-terminal domain, the a-crystallin domain. This thesis is concerned with a chloroplast localized sHsp, Hsp21. In addition to the a-crystallin domain Hsp21 contains a conserved N-terminal domain predicted to form an amphipathic a-helix with highly conserved methionine residues on the hydrophobic side. Using transgenic Arabidopsis thaliana plants that overexpress Hsp21 I found that Hsp21 improves the plant performance during concomitant heat and light stress and that Hsp21 undergoes conformational changes during stress. To characterize the different conformations, recombinant Hsp21 protein was used and different biochemical and biophysical methods as circular dichroism, fluorescence spectroscopy, mass spectrometry and site-directed mutagenesis. A similar conformational change could be induced in the recombinant Hsp21 protein by oxidation with hydrogen peroxide. In the oxidized Hsp21 protein the highly conserved methionine residues was found to be in methionine sulfoxide form. The oxidized Hsp21 showed loss in a-helical secondary structure and chaperone activity. An enzyme, peptide methionine sulfoxide reductase, was able to reduce the methionine sulfoxides and recover the chaperone activity. The present data indicate that the methionine-rich amphipathic a-helix, which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone activity very dependent on the chloroplast redox state.