Journal of Molecular Biology
Crystal Structure of the Oxidized Form of the Periplasmic Mercury-binding Protein MerP from Ralstonia metallidurans CH34
Section snippets
Redox state analysis of R. metallidurans CH34 MerP
As the reduced form of MerP can react with iodoacetamide, alkylation was used as a probe for checking the redox state of MerP. Immediately after purification, MerP was incubated with iodoacetamide. The mass spectrum of this solution (Figure 1A) shows two species with a mass difference of 114 Da corresponding to the addition of two alkylating groups. The lowest value (7519 Da) matches the theoretical molecular mass deduced from the amino acid sequence and the highest (7633 Da) matches the reduced
Conclusion
Our work describes the first crystal structure of the periplasmic mercury-binding protein MerP from R. metallidurans CH34. The architecture of the consensus sequence region in the crystallographic model of oxidized MerP is similar to oxidized superoxide dismutase chaperone and Atx1, two eukaryotic copper transporters. This model proposes that the side-chain of Y66, a conserved residue in MerP family, and the main-chain amide of C14 are good candidates to maintain C17 under its anionic form in
Bacterial strains and plasmids
The transposon Tn4378 from R. metallidurans CH34 was previously fully transferred on plasmid R1CM (ChloramphenicolR). The resulting plasmid R1CM∷Tn4378 was then used to transform a derivative of the E. coli strain CSH23 [recA1 spcR supE thisrl∷Tn10 Δpro lac]. The resulting strain E. coli CSH23 (R1CM∷Tn4378) is thus HgR, CmR, and TcR (unpublished results) and is a gift from Pr. Max Mergeay (CEN/SCK, Mol, Belgium).
Construction of expression plasmid and purification of recombinant MerP
R1CM∷Tn4378 was used as a template for PCR amplification of the merP gene. The primer
Acknowledgements
We thank David Lemaire and David Lascoux for their help in mass spectroscopy experiments, M. Mergeay for providing us the mer-containing bacteria strain and Corinne Vivès for her suggestions in the manuscript preparation. E.R. is a recipient of a PhD fellowship from the Région Rhône-Alpes.
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