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-termina l domain of the chloroplast-localized small heat shock protein, Hsp21: a se quence with highly conserved methionines, which is predicted to form an amp hipathic a-helix with the methionines. situated along one side. The functio nal role of these conserved methionines is not understood. We have found pr eviously that treatment, which causes methionine sulfoxidation in Hsp21, al so leads to structural changes and loss of chaperone-like activity. Here, m utants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-re sistant leucines. Mutants lacking the only cysteine in Hsp21 were also crea ted. Protein analyses by nondenaturing electrophoresis, size exclusion chro matography, and circular dichroism proved that sulfoxidation of the four hi ghly 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 methionin es, because it was retained after methionine-to-leucine substitution. The f unctional role of the conserved methionines in Hsp21 may be to offer a poss ibility for redox control of chaperone-like activity and oligomeric structu re dynamics.