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
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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. 280,13701-1