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| Untersuchte Arbeit: Seite: 18, Zeilen: 17-31 |
Quelle: Chapleau 2008 Seite(n): 614, Zeilen: abstract |
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| In their study, Chapleau et al. (2008) presented a novel fluorescence-based biosensor for the direct monitoring of the uptake and distribution of Hg under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, AvGFP was converted into a highly specific biosensor for this metal ion. The binding of mercury to sulfhydryl groups in proteins is extremely efficient and is likely to be the main cause for its toxicity. The mutant protein exhibited a dramatic absorbance and fluorescence change upon mercuration at neutral pH (fig. 10). Absorbance and fluorescence properties with respect to the metal concentration exhibited sigmoidal binding behavior with a detection limit in the low nM range. At very high concentrations of mercury (>200 mM), fluorescence of all tested eGFP variants vanished completely. Treatment with metal-binding agents such as β-mercaptoethanol, glutathione, or EDTA did not regenerated the fluorescence of the protein, suggesting tight binding of the metal to the protein under these conditions. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. Cysteine 205C is located in close proximity to the hydroxyl group of the chromophore tyrosine. | In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. |
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