Investigation of structural features of mutant metallothioneins: modifications in cysteine motifs of T. durum metallothionein (Mutant metallotionin’lerin yapısal özelliklerinin incelenmesi: T. durum metallotionin’in sistin motiflerinin modifikasyonu)

Metallothioneins (MTs) are characterized as low molecular weight, cysteine-rich, metal binding proteins. Since their discovery more than 40 years ago as Cd-binding proteins present in horse kidney, MT proteins and genes have been found throughout the animal and plant kingdoms as well as in the procaryote Synechococcus. The large number of cysteine (Cys) residues in MTs bind a variety of metals by mercaptide bonds [1]. Most research on protein-metal binding comes from the animal literature and little is known about the functions of the plant MTs. A novel MT (dmt) gene in Triticum durum was identified and cloned for overexpression in E.coli [2]. T. durum metallothionein (dMT) is a type 1 MT which includes three Cys-X-Cys motifs in each of the N- and C-terminal domains that are connected by a 42 residue long hinge region. This long hinge region is not observed in the mammalian homologs. dMT was overexpressed in E.coli as a GST (glutathione-S-transferase) fusion protein (GSTdMT). Both GSTdMT and dMT cleaved from GST were purified and characterized by biochemical and biophysical methods. Moreover, it was shown that GSTdMT binds 4±1 moles of Cd per one mole of protein and has a high tendency to form stable oligomeric structures [3]. In the light of these findings, the aim of the present invstigation is to determine the effect of Cys-X-Cys motifs on the metal binding capacity and affinity of dMT. The Cys motifs are modified by introducing single and double mutations to the GCSCG pattern that is observed in both β and α domains in dMT. The new mutant proteins designed to conatin CCSCG, GCSCC or CCSCC motifs. dmt gene is mutated by using site-directed mutagenesis and the mutant constructs are introduced into the pGEX4T-2 vector for expression in E. coli. The ultimate aims of this investigation are studies on structural features of mutant proteins and their metal binding properties using biophysical techniques including gel filtration chromatography, SDS- and native PAGE, dynamic light scattering, atomic absorption spectroscopy, circular dichroism spectrometry and X-ray scattering.