Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals

Autor(es)

Gabriel, Gabriele V. M. ; Viviani, Vadim R.

• Tipo de documento: Artigo Científico
• Fonte: ANALYTICAL AND BIOANALYTICAL CHEMISTRY; v. 408, n. 30, p. 8881-8893, DEC 2016.
• Citações Web of Science: 2

Resumo

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. (AU)

• Processo FAPESP: 11/23961-0 - Luciferases pH-sensitivas: origem estrutural da sensibilidade ao pH, e viabilidade de emprego em biossensores celulares de pH e cátions de metais pesados
• Beneficiário: Vadim Viviani
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/04477-9 - Desenvolvimento de biossensores raciométricos bioluminescentes de pH e metais divalentes baseados na engenharia da região sensível ao pH nas luciferases de vagalumes
• Beneficiário: Gabriele Verônica de Mello Gabriel
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/09594-0 - Análise dos transcriptomas das laternas, corpo gorduroso e túbulos de Malpighi de coleópteros, e suas relações com as origens da bioluminescência em Coleoptera e Diptera
• Beneficiário: Vadim Viviani
• Modalidade de apoio: Auxílio à Pesquisa - Regular