title: Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed.
abstract: The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Renee
authors.initials: R
authors.lastname: Dale
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ohmuro-Matsuyama
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kato
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D003198:Computer Simulation; D020128:Inhibitory Concentration 50; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry / Q000378:metabolism*; D049449:Luminescence; D008962:Models, Theoretical; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D015203:Reproducibility of Results; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D016159:Tumor Suppressor Protein p53; D000249:Adenosine Monophosphate; D049409:Luciferases, Firefly; D051736:Proto-Oncogene Proteins c-mdm2