title: Improvement of Cd(2+) uptake ability of SmtA protein by Lys/Cys mutation; experimental and theoretical studies.
abstract: The improved Cd(2+) surface affinity characteristics of a mutated cyanobacterial metallothionein SmtA (K45C) were investigated via experimental and theoretical methods. Molecular dynamics simulations were carried out using a model of Cd(2+) and other ions enclosed in a fully hydrated simulation box with the wild-type or mutated SmtA protein. The theoretical results suggested that mutated SmtA was more powerful in absorption of Cd(2+) than the wild-type protein. Then, the mutated smtA gene (from Synechococcus PCC 7942) was synthesized by simplified gene synthesis method and expressed on isopropyl-beta-d-thiogalactopyranoside induction. The protein expression was investigated by SDS-PAGE and verified by Western blotting. Finally, cadmium uptake ratio of mutant protein toward wild type was analyzed by atomic absorption. This study is the first example of cytoplasmic expression of a mutant protein. Experimental results also verified that the mutation intensifies uptake of Cd(2+) ions.
authors.affiliation: a Faculty of Science, Department of Genetics , Shahrekord University , Shahrekord , Iran.|b Biotechnology Research Institute , Shahrekord University , Shahrekord , Iran.
authors.forename: B
authors.initials: B
authors.lastname: Saffar
authors.affiliation: c Master of Science, Department of Genetics , Shahrekord University , Shahrekord , Iran.
authors.forename: A
authors.initials: A
authors.lastname: Mehri Ghahfarrokhi
authors.affiliation: d Faculty of Science, Department of Biology , Shahrekord University , Shahrekord , Iran.
authors.forename: K
authors.initials: K
authors.lastname: Mahnam
authors.affiliation: a Faculty of Science, Department of Genetics , Shahrekord University , Shahrekord , Iran.
authors.forename: M
authors.initials: M
authors.lastname: Mobini-Dehkordi
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008962:Models, Theoretical; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D002104:Cadmium; D008668:Metallothionein
keywords: SmtA metallothionein; cadmium uptake; molecular dynamics simulation; protein expression