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.
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 i...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4757408?pdf=render |
| _version_ | 1811282024488501248 |
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| author | Renee Dale Yuki Ohmuro-Matsuyama Hiroshi Ueda Naohiro Kato |
| author_facet | Renee Dale Yuki Ohmuro-Matsuyama Hiroshi Ueda Naohiro Kato |
| author_sort | Renee Dale |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T01:44:13Z |
| format | Article |
| id | doaj.art-650a93b1c3d64c9f90be47743aa767b7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T01:44:13Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-650a93b1c3d64c9f90be47743aa767b72022-12-22T03:08:05ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01112e014825610.1371/journal.pone.0148256Mathematical 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.Renee DaleYuki Ohmuro-MatsuyamaHiroshi UedaNaohiro KatoThe 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.http://europepmc.org/articles/PMC4757408?pdf=render |
| spellingShingle | Renee Dale Yuki Ohmuro-Matsuyama Hiroshi Ueda Naohiro Kato 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. PLoS ONE |
| 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. |
| title_full | 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. |
| title_fullStr | 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. |
| title_full_unstemmed | 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. |
| title_short | 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. |
| title_sort | 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 |
| url | http://europepmc.org/articles/PMC4757408?pdf=render |
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