The goal of this project is to increase the understanding of how living organisms convert chemical energy into light. Bioluminescence--the emission of light by living organisms--is a beautiful natural phenomenon that has enchanted children, challenged those who have tried to understand it, and provided the basis for an important research tool. The project addresses the need to produce novel luciferase proteins to improve current research applications of bioluminescence and to advance the development of new ones. The proposed research will be carried out at a liberal arts college with an established record of effective research training of undergraduate students. Modern facilities and equipment provide an excellent environment for students and faculty to engage in collaborative research. The PI has actively directed undergraduate research projects for 37 summers. Approximately 98 undergraduate students, approximately 70% of whom are women and approximately 10% who are from traditionally underrepresented groups, have worked with him and approximately 60% of these participants entered graduate or professional school. Students who work in the PI's lab and make substantive contributions to a project become co-authors and are actively involved in the writing of manuscripts and presenting their results at scientific meetings. The aim is to offer a meaningful research experience to a diverse group of students. Moreover, several of the students in the proposed program will be participants in a department collaborator's NSF S-STEM grant. All program participants will engage in modern mainstream bioluminescence research and will contribute positively to increasing the numbers of well-prepared graduates for entry into graduate programs and professional scientific careers.
Two major objectives are addressed in this project that were developed during related investigations with prior NSF-RUI grant support. The first is to determine the basis for the altered emission color (574 nm), resistance to low pH red shifting, and unexpectedly greater specific activity (compared to the brightest North American firefly Photinus pyralis) of a newly cloned luciferase from Photinus scintillans. The main aim is to better understand the natural determinants of firefly emission color and pH stability through mutagenesis studies of the enzymes from both species. The PI will fully characterize the physical and spectral properties of the new enzyme and undertake biochemical studies to identify key amino acid residues that determine the enhanced properties. The proposed investigation will involve a mutagenesis approach and variants of the enzymes will be studied by: steady state kinetics, quantitative analysis of bioluminescence reaction products, and spectral methods including fluorescence and bioluminescence emission techniques. With collaborator Professor Andrew Gulick, the crystal structure of a complex of the novel luciferase with DLSA, an inhibitor that binds to the active site, will be determined and the mutagenesis data will be interpreted in a structural context. The second objective is to develop new substrates and red-emitting luciferase variants for improved dual color (analyte) reporter gene applications. The plan is to seek new assay methodology with greater convenience of performance and enhanced sensitivity compared to current technology. The PI will use 2 luciferase variants and 2 bioluminescence-producing substrates-- beetle luciferin and a novel analog called benzothiophene luciferin. Additionally, the PI may explore the use of fluorine-containing derivatives of benzothiophene luciferin to be made by synthetic routes that have been designed. The 2 luciferase variants are engineered to produce well-separated green (~520 nm) and red (~617 nm) emission spectra. The PI will use several mutagenesis strategies to produce enzymes that produce light with further separated emission maxima. The assays will be optimized with respect to pH, signal stabilizers, lysing agents, substrate concentrations and spectral filters.
