A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the Nand C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS-and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 ؎ 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification. Metallothioneins (MTs)1 constitute a superfamily of ubiquitously expressed low molecular mass (6 -7 kDa), cysteine-rich proteins lacking aromatic amino acids (1). After first being identified as a cadmium-binding protein in horse kidney (2), MTs were found in a wide range of organisms from plants to fungi, and their high capacity to bind metals (e.g. copper, cadmium, zinc, mercury, and silver) in metal-thiolate clusters was demonstrated (3, 4). Several functional roles have been attributed to MTs, including heavy metal detoxification, zinc and copper homeostasis (5, 6), scavenging of reactive oxygen species (7), regulation of metalloenzymes and transcription factors (5), involvement in metabolism of metallo-drugs and alkylating agents, response to stress conditions, and the potential involvement at inflammatory sites and in apoptosis (8).Historically, MTs have been classified in three groups according to their sequence similarities. Class I consists of those with high sequence homology to mammalian MTs; class II includes all that do not display significant similarity to class I, and class III consists of phytochelatins, enzymatically synthesized peptides with a poly(␥-glutamylcysteinyl)glycine structure found in plants (9). In recent years computational analyses resulted in a more detailed classification, where the MT superfamily was divided into families, classes, and subclasses based on the location and distribution of the cysteine residues (10)...
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