Cloning, characterization and expression of a novel metallothionein gene from triticum and theoretical and experimental structure-function relationship prediction
Bilecen, Kıvanç (2003) Cloning, characterization and expression of a novel metallothionein gene from triticum and theoretical and experimental structure-function relationship prediction. [Thesis]
Metallothioneins (MTs) are small, cystein rich, low molecular mass polypeptides found in almost all organisms. They are thought to be involved in heavy-metal detoxification and metabolism of essential trace elements like copper and zinc. Unlike their mammalian counterparts, plant MTs have not been thoroughly characterized in terms of cellular regulation and function. A novel gene, from Triticum durum (pasta wheat), coding for plant MT type 1 protein was isolated and characterized. The durum mt gene was cloned in E. coli for solution X-ray scattering studies to obtain the first experimental structural data on a plant MT in the literature. Triticum durum mt gene was shown to contain 2 exons and a non-coding intron region. The coded MT protein, showing high similarity to mammalian MTs in its cystein residue distribution pattern, forms two metal binding domains bridged with an exceptionally long connecting region. This hinge region was shown to be highly conserved among plant MTs using sequence alignment algorithms on data available in the literature. Homology modeling and heuristic fragment assembly approaches were used to predict a 3D structure for the durum MT (dMT). Guided by the predicted structures, functional motif and structure searches were performed yielding a possible DNA binding and/or protein interaction function for dMT. High probability of wMT to form dimers or trimers inside the solution was also speculated. dMT was expressed in E. coli cells as a fusion protein with GST and preliminary X-ray solution scattering measurements were carried out on the purified recombinant v protein. These measurements indicated the high tendency of the protein to form aggregates in solution. Theoretical predictions and solution scattering measurements were also supported by the results of polyacrylamide gel electrophoresis and size exclusion chromatography analysis of expressed and purified recombinant dMT and GSTdMT proteins. Further, sequence and structure analyses showed a high structure and sequence similarity between dMT hinge region and the DNA binding domain of a cyanobacterial metallothionein suppressor protein (SmtB). Indeed, the results indicate that dMT metal binding domains would also bind to DNA with very high probability. These results, altogether, point to a new role for plant MTs other than metal scavenging such as being a transcription factor or a gene suppressor.
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