Biophysical and functional characterization of wheat metallothionein at molecular level

Official URL: http://192.168.1.20/record=b1225680 (Table of Contents)

Metallothioneins (MTs) are small, cysteine-rich proteins with high binding capacity for metals including Zn, Cu and Cd. MTs exist in a wide range of organisms and are classified in one super-family according to the distribution of cysteine motifs in their sequences. Plant Type 1 MTs are low molecular weight (7-8 kDa) aromatic residue lacking metal-binding proteins. They have two metal-binding domains separated by a conserved specifically long spacer region of about 30-45 amino acids. Recent studies indicate that all members of this family do not have a single unifying function; while some MTs participate in metal homeostasis others play a role in detoxification of heavy metals. Despite the large literature on MT sequences and functional roles, lack of direct biochemical and biophysical data on purified proteins hinders a comprehensive understanding of sequence, structure, metal-binding and function relationships in MTs from different sources. Due to their aggregation propensity, sensitivity to oxidation, proteolytic cleavage especially in the spacer region and difficulties in quantification, standard methods could not be directly used for purification of plant MTs. A Type 1 MT from Triticum durum, dMT, was expressed in E. coli cells as a fusion protein with GST (Bilecen et al., 2005). In the present study structure-function relationship of dMT is investigated using the fusion protein GSTdMT as a model system. The procedure developed for GSTdMT purification required strict anaerobic conditions and critical parameters including concentration of Cd and specific reducing agents, as well as choice of buffers was optimized. The purified GSTdMT was characterized by size exclusion chromatography, SDS- and native-PAGE, UV-vis absorption spectrophotometry, inductively coupled plasma optical emission spectroscopy (ICP-OES), dynamic light scattering (DLS), circular dichroism (CD), Extended X-ray Absorption Fine Structure (EXAFS) and small-angle solution X-ray scattering (SAXS). Size exclusion chromatography revealed a stable dimeric form of GSTdMT in solution. Purified GSTdMT solutions were monodisperse and homogeneous and thus suitable for structural and functional studies. The Cd binding ability of GSTdMT was initially characterized by UV-vis absorption spectroscopy and through the ICP-OES measurements the Cd2+ binding ratio was found to 3.5 Cd2+/protein. This finding was further confirmed by EXAFS measurement which strongly indicated Cd-S coordination with four sulphurs. SAXS measurements revealed that GSTdMT has an elongated shape with a radius of gyration of 3.57 nm. ab initio models resulted in a structure in which two GST molecules form an electron dense region at one end of the dimer and the two dMT molecules extend from this region. dMT structure appears to be independent of GST in the GSTdMT fusion. The combination of SAXS results with biochemical data lead to the proposal of a hairpin like model for dMT structure. Results show that the metal content and structure of dMT in the fusion protein are preserved, thus biologically relevant structural parameters can be determined using the GSTdMT construct. In a previous study (Bilecen et al., 2005) the predicted structure for the spacer region had shown similarity with that of a family that includes DNA binding proteins. In this thesis DNA binding possibility of dMT protein was examined through the whole genome PCR-based screening method. It was found that application of this method resulted in several artifacts and in our hands the method could not be used for investigation of DNA binding possibility of any protein. In part of this work response of Triticum durum cv. Balcalı-85 to environmental Cd was investigated. Balcalı-85 was subjected to increasing Cd concentrations (e.g., 0, 2, 5, 10, and 20 μM Cd). As a result, reduction in dry weight matter was observed both in roots and shoot. Also, it was found that Balcalı-85 has high capacity to retain Cd in roots. These studies were carried out as a part of an investigation which will focus on the correlation between mt-d gene expression and Cd response. In addition, southern blot analysis revealed that the mt-d gene, having 2 exons and a non-coding intron region, has a single copy in T. durum genome.