Purification and characterization of recombinant durum metallothionein domain constructs
Collak , Filiz and Dinler-Doğanay, Gizem and Yeşilırmak, Filiz and Sayers, Zehra (2008) Purification and characterization of recombinant durum metallothionein domain constructs. In: Biophysical Society Meeting 2008, Long Beach, USA
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Metallothioneins (MTs) are small proteins with high cysteine content and high binding capacity for metals like Zn, Cu and Cd. MTs exist in a wide range of organisms and are classified in one super- family according to the distribution of cysteine motifs in their sequences. Type 1 plant MTs, similar to their mammalian counterparts, have the cysteine motifs clustered in the N-and C-termini constituting the β- and -domains respectively. The two domains are connected by an unusually long (about 42 amino acids) hinge region whose structural and functional properties are unclear. Recent studies indicate that, despite the classification into a single family, all MTs do not have a single unifying function, and while some MTs help metal homeostasis others play a role in detoxification of heavy metals (e.g. Cd and As). We identified an mt gene in Cd resistant durum wheat coding for a type 1 MT (dMT) and the recombinant protein (dMT) was over-expressed in E. coli as GST fusion (GSTdMT). For detailed structural and functional investigations GST-fusion constructs of βhinge-, hinge- and the hinge-domains of dMT were over-expressed in E. coli. Proteins were purified and results of characterization by size exclusion chromatography, SDS- and native-PAGE, UV-VIS absorption spectroscopy, atomic absorption spectroscopy, dynamic light scattering, and small-angle solution X-ray scattering will be presented. Studies on the isolated domains indicate distinct metal-binding properties and structural features for these regions. GST fusion of the hinge appears to be stable reflecting an intrinsic structural organization for this domain. These findings will be discussed in terms of relevance for the native structure of dMT.
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