Development of far-red light emit thermostable luciferases of Phrixotrix hirtus for bioanalytical purposes: relation structure and function

• Grant number: 15/05949-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: November 01, 2015
• End date: February 28, 2019
• Field of knowledge: Biological Sciences - Biophysics - Radiology and Photobiology
• Principal Investigator: Vadim Viviani
• Grantee: Gabriela de Oliveira
• Host Institution: Centro de Ciências e Tecnologias para a Sustentabilidade (CCTS). Universidade Federal de São Carlos (UFSCAR). Sorocaba , SP, Brazil

Abstract

Luciferases are enzymes responsible for bioluminescence, the light emission by living organisms. They catalyze highly exergonic oxidation of a substrate - luciferin - that the energy is released in the form of light with high efficiency. Luciferases of fireflies are the better known and have a wide spectrum of light emission, usually from green to yellow. These enzymes are widely used in several biotechnological purposes, including ATP bioassay, testing with reporter genes, and cell bio imaging. However, most of the luciferases used as bio analytical tools emits light in the range of yellow-green at physiological pH, and for applications in pigmented samples and mammalian tissues red light-emitting luciferases are more appropriate. Furthermore, luciferases are generally unstable at high temperatures, so it is necessary to improve their thermostability such they could be applied as reagents and for bio imaging in mammalian tissues. The luciferase of Phrixotrix hirtus is the one that naturally produces red bioluminescence (623 nm). However, this enzyme also suffers the disadvantage of being unstable at high temperatures. The main objective of this project is to produce and select thermostable mutants of the luciferase of P. hirtus, possibly with more displaced bioluminescence spectra for the red (> 640 nm) relative to wild type luciferase, through techniques of random and site-directed mutagenesis, combinatorial chemistry and luciferin analogues. We will investigate the relationship between structure and function of these mutants obtained by molecular modeling and the determination of the three-dimensional structure by X-ray crystallography. As a secondary objective, we will try the thermal stabilization of other luciferases available in our laboratory. (AU)

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)

OLIVEIRA, GABRIELA; VIVIANI, VADIM R.. Temperature effect on the bioluminescence spectra of firefly luciferases: potential applicability for ratiometric biosensing of temperature and pH. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, v. 18, n. 11, p. 2682-2687, NOV 1 2019. (10/05426-8, 15/05949-4)

OLIVEIRA, GABRIELA; VIVIANI, VADIM R.. Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours. LUMINESCENCE, v. 33, n. 2, p. 282-288, MAR 2018. (15/05949-4, 10/05426-8)