An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe.

Lead contamination is a serious threat to human health and the environment. 2] Lead poisoning is still one of the most common environmentally caused diseases in the world today. Lead levels are typically measured by using atomic absorption spectroscopy or other related instrumental methods. Probes that can provide rapid, on-site evaluation of the lead content of a sample are very valuable for a variety of applications. A primary challenge is to construct a “turn-on” probe that responds to lead(ii) ions with a high selectivity over other heavy metal ions. This has not been achieved with chemically designed small molecules, despite some progress in the last decade. Chemical probes have been developed for the detection of other small molecules, but with limited success for heavy metal ions and paramagnetic metal ions. An evolution strategy was elegantly applied by Lu and coworkers recently to construct Pb probes based on a DNAzyme. This system is surprisingly selective towards Pb ions. We have adopted a different strategy that takes advantage of the function of metalloregulatory proteins. Nature has evolved numerous such proteins to control the concentrations of beneficial or toxic metal ions with unprecedented sensitivity and selectivity. If the biological sensory events of these proteins were reported with measurable signals, such as fluorescence, practical probes could be obtained for the detection of various metal ions. We have recently invented a method to convert the MerR-family proteins into fluorescent reporters for the detection of Hg and coinage metal ions. Herein we report the successful conversion of a novel Pb-regulatory protein in Ralstonia metallidurans CH34 into a fluorescent reporter that exhibits high selectivity and sensitivity for Pb ions. With the aid of this system, the binding of Pb ions to the Pb-regulatory protein was also characterized. A surprisingly high binding selectivity of the protein towards Pb is revealed. Ralstonia metallidurans is a Gram-negative, non-sporeforming bacillus that flourishes in millimolar concentrations of toxic heavy metals. It is the only bacterium that has been shown to contain a lead-specific resistance pathway so far. The pbr operon is a unique operon, as it combines functions involved in uptake, efflux, and accumulation of Pb ions. All the resistance genes (pbrTRABCD) in the pbr operon are regulated by the PbrR protein, which mediates Pb-inducible transcription from its divergent promoter (Figure 1). PbrR is