RUI: Site-Directed Mutagenesis Studies of Firefly Luciferase

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BranchiniMCB 9816898The long-term goal of this project is to identify the major structural features of the bioluminescent protein firefly luciferase that enable the extraordinary biochemical process leading to the emission of visible light ranging in color from blue to red. Bioluminescence--the emission of light by living organisms--is a fascinating natural phenomenon that has enchanted children, challenged those who have tried to understand it, and provided the basis for an important research tool which has been applied in ways beneficial to human health. Examples of bioluminescence can be found throughout nature in bacteria, mushrooms, jellyfish, earthworms, clams, fish and beetles, including approximately 2,000 species of fireflies. The processes by which organisms convert chemical energy into visible light are diverse from a biochemical standpoint and appear to have many evolutionary origins. Generally, a luciferase enzyme, an organic luciferin substrate, molecular oxygen and various other cofactors combine to produce light. This study is designed to identify the functions of specific active site amino acid residues within the known primary sequence of the luciferase protein from the common North American firefly Photinus pyralis. Experiments will be carried out to identify amino acids involved in substrate binding and/or catalytic function leading to the highly efficient generation of light emission in the firefly. Recombinant DNA techniques will be used to create new luciferases by substituting different amino acids for the targeted ones. Amino acids selected for mutation comprise two groups, although their functions may be interrelated. The first group of amino acid residues are very likely at or near the luciferin binding site and may be involved in bioluminescence color determination. The other group of residues are invariant among a large group of enzymes which, like luciferase, catalyze the formation of adenylates from a diverse group of carboxylic acid substrates. Preliminary work has enabled the selection of the first targets for mutagenesis studies. This study should contribute to a better understanding of the relationship between the structure of the luciferase protein and its catalytic functions. Furthermore, results so obtained will be applicable to a basic understanding of firefly bioluminescence and the fundamental process by which living organisms convert chemical energy into light. The elucidation of firefly luciferase structure-function properties is important to fundamental biochemical processes with significant practical applications extending beyond the realm of bioluminescence. Moreover, the project will be carried out at a liberal arts college providing a very positive climate for the effective training of undergraduate students. Previous bioluminescence work has been highly appealing to students and should continue to be so. All of the student and professional participants 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.