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pH-sensitive luciferases: structural origin of pH-sensitivity, and suitability of use in cell biosensors for pH and heavy metals


Beetle luciferases produce light of different bioluminescence colors, from green to red, using the same substrates D-luciferin, MgATP and molecular oxygen. Functionally, they are classified as pH-sensitive luciferases (Lampyríds), in which the bioluminescence spectrum undergoes red shift at lower pHs, presence of heavy metals and higher temperatures, and pH-insensitive luciferases(phengodids and elateríds) whose spectra are not affected by such factors. The pH-sensitive luciferases were the most studied and are actually the most used in analytical applications and as reporter genes for studies of gene expression, biosensors and cell imaging. However, the structural origin of pH-sensitivity remains unclear. Furthermore, the pH-sensitivity has often been considered an undesirable property since intracellular factors may reduce the production of green light. In 2005 we cloned a luciferase from the Brazilian firefly Macrolampis which displays a very sensitive spectrum to pH and other factors, and suggested the use of such spectral sensitivity as indicator of intracellular changes of pH and heavy metal, making the first deposit of a patent that uses firefly luciferase spectral sensitivity for analytical purposes. However, such property has not been used yet, in part because it was a ahead of time technology. Therefore, using the luciferase de Macrolampis, and other pH-sensitive luciferases cloned by our group, we aim to identify the structural determinants of pH-sensitivity, and to investigate the technological applicability of this property, developing cell and intra-cellluar biosensors for detection of pH changes and heavy metals. (AU)

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Scientific publications (5)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
VIVIANI, V. R.; AMARAL, D. T.; NEVES, D. R.; SIMOES, A.; ARNOLDI, F. G. C.. The Luciferin Binding Site Residues C/T311 (S314) Influence the Bioluminescence Color of Beetle Luciferases through Main-Chain Interaction with Oxyluciferin Phenolate. BIOCHEMISTRY, v. 52, n. 1, p. 19-27, . (12/04857-0, 11/23961-0)
GABRIEL, GABRIELE V. M.; VIVIANI, VADIM R.. Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, v. 408, n. 30, p. 8881-8893, . (13/09594-0, 11/23961-0, 14/04477-9)
GABRIEL, GABRIELE V. M.; VIVIANI, VADIM R.. Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, v. 13, n. 12, p. 1661-1670, . (11/23961-0, 14/04477-9)
VIVIANI, VADIM R.; RODRIGUES NEVES, DEIMISON; TRABUCO AMARAL, DANILO; PRADO, ROGILENE A.; MATSUHASHI, TAKUTO; HIRANO, TAKASHI. Bioluminescence of Beetle Luciferases with 6 `-Amino-D-luciferin Analogues Reveals Excited Keto-oxyluciferin as the Emitter and Phenolate/Luciferin Binding Site Interactions Modulate Bioluminescence Colors. BIOCHEMISTRY, v. 53, n. 32, p. 5208-5220, . (12/04857-0, 11/23961-0)
DE MELLO GABRIEL, GABRIELE VERONICA; YASUNO, RIE; MITANI, YASUO; OHMIYA, YOSHIHIRO; VIVIANI, VADIM RAVARA. Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, v. 18, n. 5, p. 1212-1217, . (16/15946-5, 10/05426-8, 11/23961-0, 14/04477-9, 15/22603-4)

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Filed patent(s) as a result of this research project

Solicitação em análise e dentro do prazo legal de sigilo previsto na legislação BR102017023885-7 - Universidade Estadual de Campinas (UNICAMP) . Solicitação em análise e dentro do prazo legal de sigilo previsto na legislação - November 2017, 06