Cloning expression and purification of durum wheat metallothionein domains and structural modelling
Kısaayak Çollak, Filiz (2009) Cloning expression and purification of durum wheat metallothionein domains and structural modelling. [Thesis]
Official URL: http://192.168.1.20/record=b1276327 (Table of Contents)
Metallothioneins (MTs) are small proteins with high cysteine content and high binding capacity for metals including Zn, Cu and Cd. They exist in a wide range of organisms and are classified in one super-family according to the distribution of cysteine motifs in their sequences. Type I plant MTs, similar to mammalian MTs, have the cysteine motifs clustered in the N-and C-termini constituting the β- and α-domains, respectively. In type I plant MTs the two domains are connected by a long (about 42 amino acids) hinge region whose structural and functional properties are unclear. A mt gene in Cd resistant durum wheat coding for a type I MT (dMT) was identified and the recombinant protein (dMT) was overexpressed in E. coli as GST fusion (GSTdMT) (Bilecen et al., 2005). In the present study, for detailed structural investigations; GST-fusion constructs of β-hinge, α-hinge and the isolated hinge domains of dMT were overexpressed in E. coli. Proteins were purified and were characterized by size exclusion chromatography, SDS- and native-PAGE, limited trypsinolysis, inductively coupled plasma optical emission spectroscopy (ICP-OES), UV-vis absorption spectroscopy, dynamic light scattering (DLS) and small-angle solution X-ray scattering (SAXS). GSTdMT fusion constructs were purified as stable dimeric forms in solution. Cd2+/protein ratio were found to be 1.53 ± 0.6 for GSTβ-hingedMT and 1.5 ±0.8 for GSTα-hingedMT. GSThingedMT and GSTα-hingedMT were easily cleaved from the hinge region confirming its susceptibility to proteolytic attack. Hinge region in the GSTβ-hingedMT construct, on the other hand, was protected. Cleavage was not observed in the constructs within the β- and α-domains which strongly indicated their compact structure with the bound Cd2+ ions. SAXS measurement results indicated that GSThingedMT, GSTβ-hingedMT and GSTα-hingedMT have symmetric, elongated shapes. ab initio models revealed structures with GST molecules forming electron dense regions at the center of mass and the partner dMT domains extending from this region. Evidence from Cd-binding, tryptic cleavage and SAXS models suggest that hingedMT, β-hingedMT and α-hingedMT structures fold independently of GST in the fusion constructs. The combination of SAXS results with biochemical data indicated extended structures for the dMT domains and supports the dumbbell model previously proposed for durum wheat MT (Bilecen et al., 2005).
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