Durum wheat metallothionein mutants and their biophysical characterization
Saygı, Ceren (2010) Durum wheat metallothionein mutants and their biophysical characterization. [Thesis]
Metallothionein (MT) proteins are characterized as low molecular weight, cysteine (Cys)-rich, metal binding proteins that were discovered more than 50 years ago as Cd-binding proteins present in horse kidney. They have been found in wide range of organisms and their classification was based on the phylogenetic relationships and patterns of distribution of Cys residues along the MT sequences (Binz & Kagi, 2001). They bind a variety of metals with d10 electronic configurations through mercaptide bonds with Cys residues (Vasak & Hasler, 2000). In the present study effects of mutations of Cys residue distributions on the metal-binding properties of a Cd-binding type 1 MT from Triticum durum are investigated. For this purpose, modifications were introduced to the cys-motifs of the native durum MT, dMT. Double (G61CG65C) and single mutants (G8C, G12C, G61C, G65C) were produced by site-directed mutagenesis Based on results from earlier work (Bilecen et al., 2005) mutants were expressed in E. coli as glutathione-S-transferease (GST) fusion proteins. Proteins were purified and characterized by affinity and size exclusion chromatography, SDS- and Native-PAGE, limited trypsinolysis, inductively coupled plasma optical emission spectroscopy (ICP-OES), UV-Vis absorption spectroscopy, circular dichroism spectropolarimetry (CD), dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS). Expression of the mutants G8C, G12C, G61C and G65C was stable and the proteins were purified for biophysical characterization. Expression of the double mutant G61CG65C, on the other hand, could not be detected in the soluble E. coli fractions and efforts to locate it in inclusion bodies also failed. All the over-expressed mutants were purified as homodimers in solution. Protein yield for the mutant preparations ranged between 10 to 15 mg per liter of bacterial culture. The UV-Vis absorption spectra for all the mutants displayed the typical shoulder at 250 nm indicating Cd-binding. The G8C, G12C and G61C mutants had a Cd2+ to protein ratio of 3.5±1 which is similar to that observed with native GSTdMT. The G65C, however, showed enhanced Cd binding with a ratio of 4.4 Cd2+ per protein, thus binding an additional Cd for each mole of protein compared to native GSTdMT. Proteolytic cleavage results of the G65C indicated that this mutant has more compact structure compared to native GSTdMT. Shape models obtained from SAXS data of G61C showed that the shape envelope of this mutant is similar to that of the native GSTdMT. G65C mutant obtained in during these studies offers a possibility for investigation of the Cd-binding mechanisms of MTs and for designing MTs that can be used in applications including biosensors. Future work is needed to understand the basis of enhanced Cd-binding capacity and to determine if this property is preserved when the protein is cleaved from its GST partner.
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