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.
Highlights
The firefly luciferase complementation assay (FLCA) is an assay that detects protein-protein interactions in vitro, in cellulo, and in vivo [1, 2]
We found that the change in luminescence kinetics between the FLCA and full length luciferase is due primarily to the rapid dissociation of the protein pair fused to NFLuc and CFLuc, a lower adenylation rate, and an increased affinity of the NC complex to LH2AMP
The model constructed in this study demonstrates that the relationship between luminescence and the affinity of the protein pair fused to NFLuc and CFLuc is exponential rather than linear
Summary
The firefly luciferase complementation assay (FLCA) is an assay that detects protein-protein interactions in vitro, in cellulo, and in vivo [1, 2]. The assay detects the bioluminescence that is emitted during the oxidation of the substrate, D-luciferin (LH2). The N-terminal and C-terminal domain of firefly luciferase (NFLuc and CFLuc, respectively) are genetically fused to a protein pair of interest via a linker peptide [3]. Mathematical Model of Luciferase Complementation each other, NFLuc and CFLuc reconstitute the active site of the enzyme. This results in emission of luminescence when LH2 is added in the reaction
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