Abstract
In this work, we have analyzed both at stoichiometric and at conformational level the CdII-binding features of a type 2 plant metallothionein (MT) (the cork oak, Quercus suber, QsMT). To this end four peptides, the wild-type QsMT and three constructs previously engineered to characterize its ZnII- and CuI-binding behaviour, were heterologously produced in Escherichia coli cultures supplemented with CdII, and the corresponding complexes were purified up to homogeneity. The CdII-binding ability of these recombinant peptides was determined through the chemical, spectroscopic and spectrometric characterization of the recovered clusters. Recombinant synthesis of the four QsMT peptides in cadmium-rich media rendered complexes with a higher metal content than those obtained from zinc-supplemented cultures and, consequently, the recovered CdII species are nonisostructural to those of ZnII. Also of interest is the fact that three out of the four peptides yielded recombinant preparations that included S2−-containing CdII complexes as major species. Subsequently, the in vitro ZnII/CdII replacement reactions were studied, as well as the in vitro acid denaturation and S2− renaturation reactions. Finally, the capacity of the four peptides for preventing cadmium deleterious effects in yeast cells was tested through complementation assays. Consideration of all the results enables us to suggest a hairpin folding model for this typical type 2 plant CdII-MT complex, as well as a nonnegligible role of the spacer in the detoxification function of QsMT towards cadmium.
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Acknowledgements
This work was supported by the Spanish Ministerio de Ciencia y Tecnología grants BIO2006-14420-C02-01 for S.A., BIO2006-14420-C02-02 for M.C. and AGL2003-00416 for M.M. G.M. and R.O. received predoctoral fellowships from the Pla de Formació de Personal Investigador del DURSI, Generalitat de Catalunya, and the Departament de Química, Universitat Autònoma de Barcelona, respectively. We especially want to acknowledge technical support from Roger Bofill and fruitful scientific discussions with Armida Torreggiani. We also thank the Serveis Científico-Tècnics de la Universitat de Barcelona (GC-FPD, ICP-AES, ESI-MS) and the Servei d’Anàlisi Química de la Universitat Autònoma de Barcelona (CD, UV–vis) for allocating instrument time.
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Domènech, J., Orihuela, R., Mir, G. et al. The CdII-binding abilities of recombinant Quercus suber metallothionein: bridging the gap between phytochelatins and metallothioneins. J Biol Inorg Chem 12, 867–882 (2007). https://doi.org/10.1007/s00775-007-0241-y
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DOI: https://doi.org/10.1007/s00775-007-0241-y