Engineering Amydetes vivianii luciferase for use as bioanalytical reagent and as metal biosensor

• Grant number: 18/26151-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2019
• End date: June 30, 2021
• Field of knowledge: Biological Sciences - Biophysics - Radiology and Photobiology
• Principal Investigator: Vadim Viviani
• Grantee: Gabriel Felder Pelentir
• Host Institution: Centro de Ciências e Tecnologias para a Sustentabilidade (CCTS). Universidade Federal de São Carlos (UFSCAR). Sorocaba , SP, Brazil
• Associated research grant: 10/05426-8 - Arthropod bioluminescence: biological diversity in Brazilian biomes, biochemical origin, structural/functional evolution of luciferases, molecular differentiation of lanterns, biotechnological, environmental and educational applications, AP.TEM

Abstract

Lightbug luciferases emit light in the yellow-green region and their spectra are sensitive to pH and heavy metals. In recent decades firefly luciferases have been extensively used in ATP bioanalyses, cell viability, enzyme assays, and their genes have been employed as the most popular reporter genes for studying gene expression, biosensors and bioimaging. Recently, our group identified the metal binding site and mechanism of pH sensitivity in these luciferases, allowing engineering of binding sites and initiating the development of luciferases with different spectral sensitivities to different metals. Amydetes vivianii luciferase is the luciferase with the most shifted spectrum for blue (539 nm) among pH-sensitive luciferases, exhibiting the lowest pH sensitivity. For this reason, this enzyme provides a good starting point for developing more specific metal and pH sensor luciferases. Thus, in this project we will perform site-directed mutagenesis of the pH sensing region, characterize the sensitivities to pH and different metals such as cadmium and mercury, the kinetics of these mutants, and finally we will obtain standard ATP curves to use these luciferases for simultaneous analysis of intracellular ATP and metal concentration.

Scientific publications

(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)

PELENTIR, G. F.; BEVILAQUA, V. R.; VIVIANI, V. R.. A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, v. 18, n. 8, p. 2061-2070, AUG 1 2019. (10/05426-8, 15/06875-4, 18/26151-9)