Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.
Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.
Laboratory of Biochemistry and Biotechnology of Bioluminescence, Department of Physics, Chemistry and Mathematics, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Rodovia João Leme dos Santos, SP-264, km 110, Sorocaba, São Paulo, 18052780, Brazil.|Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Rodovia Washington Luís, km 235, São Carlos, São Paulo, 13565-905, Brazil.
Gabriele V M
GV
Gabriel
Laboratory of Biochemistry and Biotechnology of Bioluminescence, Department of Physics, Chemistry and Mathematics, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Rodovia João Leme dos Santos, SP-264, km 110, Sorocaba, São Paulo, 18052780, Brazil. viviani@ufscar.br.|Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Rodovia Washington Luís, km 235, São Carlos, São Paulo, 13565-905, Brazil. viviani@ufscar.br.|Graduate School of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Sorocaba, São Paulo, 18052780, Brazil. viviani@ufscar.br.
Vadim R
VR
Viviani
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002413:Cations, Divalent; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D048888:Fireflies / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008628:Mercury / Q000032:analysis*; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000032:analysis*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000032:analysis*
D002413:Cations, Divalent; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D009532:Nickel; D049409:Luciferases, Firefly; D008628:Mercury; D015032:Zinc
Luciferases; Metal binding sites; Metal biosensor; Ratiometric curve; Reporter gene; pH sensitivity