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.
News published in Agência FAPESP Newsletter about the scholarship: