Comparative Characterization of Apo-, Reconstituted- and In Vivo-Folded forms of a Durum Wheat Metallothionein

Durum wheat metallothionein (dMT), a plant type 1 metallothionein, with a long “hinge” region between metal coordinating cysteine clusters, is efficient cadmium (Cd) chelator. In this work, biophysical features of purified recombinant holo-dMT, its demetallated form (apo-dMT) and the reconstituted Cd5-dMT are compared to obtain insight into the structure and metal binding features of this protein. Results show that the purified holo-dMT is polydisperse and has 5.3±0.5 Cd2+ ions per molecule. Demetallation followed by size exclusion chromatography yields homogeneous apo-dMT which can be reconstituted with Cd2+. Synchrotron small angle X-ray scattering (SAXS) demonstrates that apo-dMT, at pH 2.0, is flexible and extended in solution. According to UV-vis, CD and native-PAGE data conformation of apo-dMT is sensitive to pH changes in the range 2.0 to 8.0. Reconstitution of the apo-protein at pH 8.0, with Cd2+ appears to take place in two phases during which first the monomer is folded to accommodate 5 Cd2+ ions and then reorganization into oligomeric forms allows incorporation of further metal ions. SAXS data indicate that holo-dMT has limited flexibility in structure, but its conformation is significantly more compact than that of apo-dMT. Results of UV-vis and circular dichroism spectroscopy show that the in vitro folded protein is structurally different from the purified holo-dMT with the same number of Cd2+ ions.This work was supported by Sabanci University Internal Research Fund Project No. IACF08-00514 and by the bilateral program TUBITAK-Julich Research Center projects TBAG-U-155 and TBAG-U-157.