Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity

Abstract

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.

Fig. 1.

Nondenaturing PAGE of wild-type and mutant Hsp21 showing oxidation-dependent structural changes of the Hsp21 oligomer. The mutants with methionines substituted by leucine, Hsp21-M(49,52,55,59)L and Hsp21-M(49,52,55,59,62,67)L, are referred to as -4M, and -6M. The mutants with cysteine replaced by alanine, Hsp21-C151A and Hsp21-M(49, 52,55,59,62,67)L,C151A, are referred to as -C and -6M-C.

Fig. 2.

Size-exclusion chromatography of wild-type and mutant Hsp21 showing oxidation-dependent structural changes of the Hsp21 oligomer. Chromatograms of (A) control samples and (B) oxidized (5mM H₂O₂) samples. The asterisk in the upper right panel indicates the 100 kD form of Hsp21 after oxidation in ammonium bicarbonate buffer. The mutants with methionines substituted by leucine, Hsp21-M(49,52,55,59)L and Hsp21-M(49,52,55,59,62,67)L, are referred to as -4M, and -6M. The mutants with cysteine replaced by alanine, Hsp21-C151A and Hsp21-M(49,52,55,59, 62,67)L,C151A, are referred to as -C and -6M-C.

Fig. 3.

High-temperature-induced aggregation of wild-type and mutant Hsp21 oligomers. Nondenaturing PAGE after incubation of wild-type and mutant Hsp21 at temperatures indicated in the figure for 1 h. The mutants with methionines substituted by leucine, Hsp21-M(49,52,55,59)L and Hsp21-M(49,52,55,59,62,67)L, are referred to as -4M, and -6M. The mutants with cysteine replaced by alanine, Hsp21-C151A and Hsp21-M(49,52,55,59,62,67)L,C151A, are referred to as -C and -6M-C.

Fig. 4.

CD spectra showing oxidation-induced loss of α-helical signal. Solid line: control protein samples. Dashed line: protein samples oxidized with 5 mM hydrogen peroxide. (A) Wild-type Hsp21. (B) Hsp21 mutant with four methionines substituted by leucine, Hsp21-M(49,52,55,59)L, is referred to as -4M; C Hsp21 mutant with cysteine replaced by alanine, Hsp21-C151A, is referred to as -C. The arrows point out the CD signal at 222 nm, indicative of α-helical secondary structure. The change in CD upon oxidation is significant, giving upon oxidation of the Hsp21 wild type, on average, a 20.2% decrease at 222 nm in four independent experiments with a standard deviation of 2.2%. For the mutant Hsp21 proteins with either four or six methionines replaced by leucines, there is no change at 222 nm upon oxidation.

Fig. 5.

Light-scattering measurements of the chaperone-like activity of wild-type and mutant Hsp21 with citrate synthase (A) or insulin (B) as substrates. Aggregation of citrate synthase and insulin with no chaperone added is indicated in the figure as a negative control, and the suppression of aggregation by the chaperone-like activity of αB-crystalline (Horwitz 1992; Lindner et al. 1997) is indicated in the figure as a positive control. sHsps do not show any increase in light scattering without citrate synthase and insulin (data not shown). Standard deviation bars are indicated for incubations with wild-type and mutant Hsp21. Hsp21 mutant with six methionines substituted by leucine, Hsp21-M(49,52,55,59,62,67)L, is referred to as -6M; Hsp21 mutant with cysteine replaced by alanine, Hsp21-C151A, is referred to as -C. The insert in panel A shows the chaperone-like activity of control or oxidized -6M mutant Hsp21.

Fig. 6.

Conformational stability of Hsp21 oligomer studied by urea gradient electrophoresis and tryptophan fluorescence. (A) Urea gradient polyacrylamide gel showing the intact wild-type pea (Pisum sativum) Hsp21 oligomer (I) and how the oligomer disassembles into monomers at higher urea concentrations (II). At even higher urea concentrations (III), unfolding of the Hsp21 monomers leads to a decrease in electrophoretic mobility. (B) The total fluorescence (calculated by integration of fluorescence between 315 and 420 nm) of wild-type pea (P. sativum) Hsp21 is presented for increasing urea concentrations.

Fig. 7.

Tryptophan fluorescence of wild-type and mutant Hsp21 in increasing urea concentration. The total fluorescence (calculated by integration of fluorescence between 315 and 420 nm) is presented for increasing urea concentrations. Data were normalized by setting the fluorescence value in 0 M urea for each protein to 100%. Filled circles: wild-type Hsp21. Filled squares: Hsp21 mutant with four methionines substituted by leucine, Hsp21-M(49,52,55,59)L, is referred to as -4M. Filled triangles: Hsp21 mutant with six methionines substituted by leucine, Hsp21-M(49,52,55,59,62,67)L, is referred to as -6M.