Biosynthesis of Triplet States via Luciferase

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Michael C Pirrung, Christopher J Bardeen both of the Department of Chemistry and Jiayu Liao of the Department of Bioengineering at the University of California-Riverside are studying the use of a bioluminescent system of the firefly to generate high energy states. Such states have unique chemical reactivity in biological systems and can convert ordinary oxygen into a species that kills cells.

Because the system depends on a protein that can be genetically engineered, cell killing can be controlled as a dynamic response to conditions. This tool could, in principle, be exploited in synthetic biology and genetic logic circuits. Fundamental science will be advanced because no controlled means to generate such high energy states biologically is currently available.

This project will benefit society via the provision of interdisciplinary training for students and the broadening of participation by groups underrepresented in science.

This project will focus on using bioluminescence to efficiently generate triplet excited states, a process usually achieved for biological chromophores by photoexcitation. This research will fulfill a need for the controlled biological creation of singlet oxygen, a reactive oxygen species (ROS) involved in oxidative stress and the immune response. The investigators will achieve this goal by developing pairs of luciferase mutant proteins and synthetic luciferin analogs that can generate triplet states and, via sensitization, singlet oxygen, a species otherwise difficult to produce with biological / genetic control.

Unnatural luciferin-luciferase pairs will be useful to modify biology by ablation of specific cells in vitro or in vivo based on gene expression of luciferase mutants. These goals will involve generation of non-native luciferase substrates, whose acceptance by wt firefly luciferase is typically much lower than luciferin. To maximize utility of these analogues, modification of the enzyme using protein engineering techniques will be undertaken.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.