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Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

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Espart, Anna y Marín, Maribel y Gil Moreno, Selene y Palacios, Óscar y Amaro Torres, Francisco y Martín González, Ana María y Gutiérrez Fernández, Juan Carlos y Capdevilla, Mercé y Atrian, Sílvia (2015) Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins. International Journal of Biological Sciences, 11 (4). pp. 456-471. ISSN 1449-2288

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The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, in-cluding 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tet-rahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been in-ternal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and op-timization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).


Tipo de documento:Artículo
Palabras clave:Metallothionein, Functional Differentiation, Metal specificity, Zinc, Copper, Tetrahymena thermophila.
Materias:Ciencias Biomédicas > Biología
Ciencias Biomédicas > Biología > Invertebrados
Ciencias Biomédicas > Biología > Microbiología
Código ID:42342
Depositado:25 Abr 2017 14:30
Última Modificación:26 Abr 2017 07:29

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