Wild-type NikR binds to its promoter DNA in the presence of Ni2+ ions, and a number of other divalent metal ions such as Cu2+, Zn2+, Co2+, Mn2+, and Cd2+ can also induce protein-DNA complex formation. However, NikR does not bind to DNA in the presence of 50 μM UO22+. The <triple> mutant NikR′ binds to DNA neither in the absence of metal ions nor in the presence of Ni2+ ions, but it forms a protein-DNA complex in the presence of UO22+. In comparison to NikR, the metal selectivity of NikR′ has been altered. Experiments with other metal ions show that the mutant protein only forms the protein-DNA complex in the presence of the uranyl cation while Ni2+, Zn2+, Co2+, Cu2+, Cd2+, Mn2+, and Fe2+ ions do not result in any observable complex formation. Attempts to load NikR with uranyl or NikR′ with Ni2+ did not yield any observable metal binding. Thus, this mutant NikR′ shows a uranyl-specific DNA-binding ability.
Wednesday, September 30, 2009
“Uranyl ion” is the traditional name of dioxidouranium(2+), [UO2]2+. According to Wikipedia, [UO2]2+ is “the most common species encountered in the aqueous chemistry of uranium”. Wegner et al. designed a uranyl-selective DNA-binding protein using the template of NikR, a nickel-dependent transcriptional repressor from E. coli. The binding site of wild-type NikR was modified by a series of mutations (Val72Ser, His76Asp and Cys95Asp) to introduce extra hard equatorial ligands to favour binding of [UO2]2+.