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   PbrR-based lead bioabsorbent

         Project > Expansion > LeadBioabsorbent
    After the completion of MerR-based mercury decontamination kit, we decided to take another example to verify the validness of our design for metal binding peptide. Based on the previous homology study of MerR family proteins, we took a lead-sensing protein, PbrR, as the second research target.

    Lead contamination is a serious threat to human health and the environment. Lead poisoning is still one of the most common environmentally caused diseases in the world today.[1] As the concentration of such toxic ions is generally low, which present a huge challenge for environmental engineers to both detect and to absorb the pollutant with traditional chemical methods. A revolutionary strategy was taken into consideration, which took the advantage of metalloregulatory proteins with capability of sensing and absorbing the Pb(II) ions.

Sequence alignment of MerR and PbrR
Figure 1 Sequence alignment of MerR and PbrR. MerR family TFs share a highly conserved homology at their metal binding domains (Brown et al., 2003; Hobman, 2007), which implies that our strategies of bioabsorbent engineering might be applicable to other cases of heavy metals.

    Nature has evolved numerous such regulating proteins to control the concentrations of beneficial or toxic metal ions with extraordinary sensitivity and selectivity.[1] As is known, the MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognized.[2] The majority of regulators in the family respond to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. A subgroup of the family activates transcription in response to metal ions. This subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region. PbrR is a MerR family protein found in Ralstonia metallidurans CH34, a bacterium specifically adapted to survive under toxic heavy metal environment. The PbrR protein is responsible for regulation of lead(II) efflux pumps involved in lead detoxification inside R. Metallidurans.

    Due to the highly conserved homology of protein MerR and PbrR (Fig.1), we were able to apply the strategy used for MerR engineering to the development of lead bioabsorbent. Based on the crystal structure study of MerR, the metal binding domain of PbrR was recognized by sequence alignment with MerR.(Fig 2) [3] Further, 3D structure was also conducted.(Fig3)

Pb binding domain predicted by sequence alignment.
Figure 2 Pb binding domain predicted by sequence alignment. Three Cys-residues located at 79,114,123 specifically binds Pb(II) ions by forming a metal binding pocket.

Figure 3 3D structure modeling of PbrR. Note that PbrR comprises 2 domains, a metal binding domain at the C terminal and a DNA binding domain at the N-terminal, joined together by a interface domain.

    Then we designed PbrR metal binding peptide, with consisted of two tandem duplications of α-helix 5 linked by a flexible linker, SSG, and followed by a short peptide sequence.(Fig 4) These direct tandem α-helices fold back on each other into an antiparallel, coiled-coil hairpin. Three Cys-residues located at 79,114,123 specifically binds Pb(II) ions by forming disulfide bonds within the engineered dimer.

Design of PbrR metal binding peptide and structure prediction
Figure 4 Design of PbrR metal binding peptide and structure prediction. Within the engineered dimer, six Cys-residues centralize 2 metal binding pocket, each of which specifically binds Pb(II) ions by forming disulfide bonds .


[1] Peng Chen, Bill Greenberg, Safiyh Taghavi, Christine Romano, Daniel van der Lelie, and Chuan He, An exceptionally selective lead (II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead (II) probe, Angew. Chem. 117, 2005, 2775 –2779.
[2] Nigel L. Brown, Jivko V. Stoyanov, Stephen P. Kidd, Jon L. Hobman, The MerR family of transcriptional regulators, FEMS Microbiology Reviews, 27, 2003, 145-163.
[3] Lingyun Song, Jonathan Caguiat, Zhongrui Li, Jacob Shokes, Robert A. Scott, Lynda Olliff, and Anne O. Summers, Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site, Journal of Bacteriology, 186(6), 2004, 1861–1868.

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