Functional and structural investigations of a metallothionein from durum wheat, dMT
Dinler-Doğanay, Gizem and Dede, Filiz and Kısaayak Çollak, Filiz and Sayers, Zehra (2007) Functional and structural investigations of a metallothionein from durum wheat, dMT. In: Protein Society 21st Symposium, Boston, USA
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Plant metallothioneins (MTs) are low molecular weight (7- 8 kDa), sulphydryl-rich, aromatic residue lacking metalbinding proteins which have two terminal metal-binding clusters separated by a conserved distinctively long hinge region of about 50 amino acids. The length and conserved sequence of the hinge region distinguishes plant MTs from their mammalian counterparts, suggesting additional roles other than metal binding and detoxification. Homology modeling indicates mammalian-like folds for the metalbinding domains of dMT, and ab initio calculations yield a DNA-binding like structural motif for the hinge region1. Here, we study the structural features of full-length dMT Abstracts 263 and its constructs in unstructured, metal-free (apo) and metal-bound (holo) states using various biophysical and biochemical characterization techniques and we investigate putative DNA-binding interactions of the hinge region using functional assays (e.g. whole-genome PCR technique) for the apo- and holo-protein. The Cd-binding dMT is expressed in Escherichia coli as a Glutathione-STransferase fusion protein and cleaved from this tag for further analyses. The exact Cd-bound state of dMT and oxidation of thiols are kept under control through preparation of apo-protein from purified Cd-dMT prior to reconstitution by titration of equivalent amounts of Cd solution under anaerobic conditions. 4 mole equivalence of Cd could saturate dMT as followed by spectral changes of circular dichroism and UV-Vis absorption spectroscopy. This finding is further confirmed by parallel inductively coupled plasma optical emission spectroscopy measurements. Small angle X-ray solution scattering measurements and gel-filtration chromatography revealed an oligomeric form for the Cd-bound dMT whereas apo dMT is monomeric as also shown by native state electrospray ionization mass spectrometry experiments. Taken together, our results suggest that structural rearrangements driven by metal-binding result in oligomerization/complex formation that may lead to functional variations. Acknowledgements This work is supported by a joint TUBITAK and JULICH grant, TBAG-U-157 105T535. 1. Bilecen et al., 2005, J. Biol. Chem. 280,13701-11
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