Plant type 1 metallothioneins: candidates for intrinsically unstructured proteins
Dinler-Doğanay, Gizem and Yeşilırmak, Filiz and Yar, Sümeyye and Collak, Filiz and Sayers, Zehra (2008) Plant type 1 metallothioneins: candidates for intrinsically unstructured proteins. In: Biophysical Society Meeting 2008, Long Beach, USA
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Type 1 plant metallothioneins (MTs) are low molecular weight (7–8 kDa), sulphydryl-rich, aromatic residue lacking, metal-binding proteins with two terminal metal-binding clusters separated by a long hinge region of about 50 amino acids. The length and conserved sequences in the hinge region distinguish type 1 MTs from their mammalian counterparts, suggesting additional roles other than metal binding and detoxification. Homology modeling of a Cd-binding durum wheat MT (dMT) indicates mammalian-like folds for the metal-binding domains, and ab initio calculations yield a DNA-binding like structural motif for the hinge region. Here, we study the structural features of full-length dMT in metal-free (apo) and metal-bound (holo) states using various biophysical and biochemical techniques. Cd-bound dMT is expressed in Escherichia coli as a GST fusion protein and cleaved from this tag for further analyses. Small angle X-ray solution scattering measurements and gel-filtration chromatography revealed holo-dMT as a dimer whereas apo-dMT is monomeric as shown by native state mass spectrometry experiments. Far-UV circular dichroism (CD) measurements revealed apo- and holo-dMT as a random coiled protein. Near-UV CD measurements showed two maxima at 246.6 and 266 nm for holo-dMT. The amount of bound Cd was determined as 5±1 M equivalent by inductively coupled plasma optical emission spectroscopy. When Cd metals were stripped, the two CD maxima disappeared and this form of dMT showed CD spectrum similar to that of the acid unfolded dMT, suggestive of disordered structure for the apo form. Interestingly, Kratky plot of the SAXS data lacks the characteristic maximum observed for globular proteins, indicating that Cd-bound dMT is in a molten globule like conformation. Taken together, our results put forward the possibility of dMT as a candidate for an intrinsically unstructured protein.
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