The conserved methionines in the chloroplast small heat shock protein - Role in chaperone-substrate interactions and effects of methionine sulfoxidation on chaperone activity
Sammanfattning: The chloroplast-localized small heat shock protein (sHsp) Hsp21 belongs to the family of alpha-crystallin like sHsps which form large oligomeric structures and protect partly unfolded aggregation-prone proteins against aggregation. The main focus for this thesis has been on the highly conserved methionines in Hsp21. These methionine residues are situated on one side of an amphipathic alpha-helix. Methionines are easily oxidized which leads to dramatical changes in the biophysical properties of the methionine side chain. The dependence of the conserved methionines for Hsp21 chaperone-like activity has been studied and the impact of methionine sulfoxidation on Hsp21 structure and function. Direct analysis of the methionione sulfoxide content of Hsp21 was performed by MALDI/TOF mass spectrometry. The methionine sulfoxidation was found to occur concomitantly with a change in the oligomeric conformation and loss of chaperone-like activity, both probably due to loss of alpha-helical structure in the methionine-rich region. By construction of Hsp21 mutants it was concluded that leucines can replace methionines in terms of chaperone-like activity. Furthermore, it was discovered that methionine sulfoxides in Hsp21 were reducible by a chloroplast-localized form of peptide methionine sulfoxide (pPMSR) and Hsp21, with its many oxidizable methionines is thus a likely endogenous substrate for pPMSR. Hence, pPMSR provides a means for keeping the methionines in Hsp21 in their reduced state and therby protection of Hsp21 chaperone-like activity in vivo. By using peptide library arrays map the sites of interaction between the methionine-rich region in Hsp21 and at least two different regions in the model substrate protein citrate synthase (CS), probably reflecting a differential chaperone-like activity of Hsp21. One of these regions belongs to a surface exposed alpha-helix in porcine CS. This alpha-helix is absent in CS from the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus. The data presented in this thesis point to a role of the methionines in substrate binding, that pPMSR co-evolved with Hsp21, that methionines may be preferred over leucines due to structural flexibility and efficient subunit exchange and that methionine sulfoxidation may play a role in regulation.
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