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. Examples of bioluminescence can be found throughout nature in bacteria, mushrooms, jellyfish, earthworms, clams, fish and beetles. This project addresses the relationship between the structure of the enzyme firefly luciferase and its detailed role in the catalysis of two sequential reactions that ultimately lead to the production of yellow-green light in the North American firefly, Photinus pyralis. New luciferases made by recombinant DNA techniques will be evaluated by spectroscopic and kinetics methods to examine the effects of mutations on each reaction. This approach may also provide evidence of the role of essential conformational changes in the luciferase structure. The results of this research will enhance the basic understanding of firefly bioluminescence and the fundamental process by which living organisms convert chemical energy into light. 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. Broader Impacts. This 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 principal investigator has actively directed undergraduate research projects for 32 years. Approximately 85 undergraduate students, the majority of whom are women and minorities, have worked with him, and an estimated 70% of these participants have entered graduate or professional school. The aim of this project is to offer a meaningful research experience to a diverse group of students in a multicultural environment. All student and professional participants in this project will be involved 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. Results of this research may lead to improvements in the uses of firefly luciferase, for example, in the detection of food contaminated with bacteria or as a reporter of gene expression and regulation.
Our laboratory very successfully investigated the major objectives of this NSF-RUI award. Over the course of the entire grant period that ended in November, 2013, we acknowledged the NSF support in 11 refereed journal articles, an invited review article and a patent "Isolated Luciferase gene of L. italica" issued in the USA and Europe. Major highlights included: a Journal of the American Chemical Society communication; a Biochemistry rapid report; the participation of 6 undergraduate co-authors; 8 international and/or domestic collaborations; and 10 oral/poster presentations. Our research focused on improving the basic understanding of how the enzyme luciferase from fireflies catalyzes reactions that convert chemical energy into light, a process called bioluminescence. Two highly significant accomplishments during this grant period were: (1) the chemical trapping of an elusive firefly luciferase protein conformation in which bioluminescence occurs; and (2) collaborative crystallographic studies with Professor Gulick (Hauptman Woodard Institute, Buffalo, New York). Together publications describing these projects provided biochemical and structural evidence that firefly luciferase functions according to the domain alternation mechanism. We produced crystal structures of the luciferase protein in two major conformations related by an ~140° rotation of the small C-terminal domain about the large N-terminal domain. Our biochemical evidence showed that adenylation of substrate firefly luciferase by ATP and the subsequent light producing oxidation of the adenylate intermediate occur in two separate active sites represented by the major crystallographic conformations. The key to obtaining the structure of the elusive oxidation conformation was the development of a protein variant with surface cysteine residues that were covalently cross-linked, thereby locking the protein into a conformation not previously observed. These results are quite significant, as the luciferases have now been structurally related to an important large superfamily of enzymes that includes human metabolic proteins. This first set of beetle luciferase crystal structures representing the 2 major conformations should enable us and other researchers to use modeling methods to further investigate luciferase and superfamily enzyme function. We expect that this will lead to the development of novel luciferase tools for biochemical research applications. To address another objective, we made a chimeric enzyme, which contained the N-terminal domain residues 1-439 of P. pyralis luciferase and the C-terminal domain residues 442-548 of L. italica luciferase. In evaluating the properties of this new protein, we made the serendipitous discovery that the chimeric enzyme possessed specific activity, total light output, and stability that were significantly enhanced relative to both wild-type enzymes. The 2-fold increase in specific activity (light output) is due in part to changes in the rates of the enzyme-catalyzed reactions, and the new chimeric protein is a fundamentally improved luciferase enzyme. It should be possible to develop novel variants of the chimeric enzyme to improve the sensitivity of bioanalytical applications that are based on measuring light emission. Additionally, in collaboration with Promega Biosciences, 5 luciferin analogs were designed, synthesized and thoroughly evaluated with P. pyralis luciferase. One of these new compounds, a benzothiophene-substituted luciferin, produced long-lived and bright blue-shifted bioluminescence. We believe that the unusual properties of the analog should provide greater sensitivity in a variety of applications including those designed to monitor genetic activity. The broader impacts of this award include the research participation of 14 undergraduates, including 8 women and 5 NSF S-STEM Science Leaders (3 of whom are students of color). Eight summer research stipends were provided to undergraduates. The undergraduate participants contributed 6 co-authorships and 3 independent presentations at scientific meetings. Two of the students who co-authored papers won prestigious Goldwater scholarships. After graduation the student participants have entered doctoral programs in biochemistry, neuroscience and chemistry, medical school, and science teaching at a local high school. One former undergraduate wrote an invited review article with me, taught English for a few years in Japan, worked in a DNA sequencing facility at a biomedical research institute and is now applying to doctoral programs in several areas of biochemistry. Also, one undergraduate student did a joint project in Professor Gulick’s laboratory at the Hauptman Woodward Institute in Buffalo, New York. During the spring 2013 semester, 2 high school students from a local magnet school joined the lab (after school hours) for approximately 2 months to do their senior year culminating science projects. We have created a team environment with the research technicians and students participating in weekly meetings and social events including a trip to the American Museum of Natural History exhibit "Creatures of Light". We have updated and maintained our bioluminescence web site (http://BRG.conncoll.edu) and provided luciferase plasmids and expertise to approximately 40 requesting scientists.
