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Development of an optimized backbone of FRET biosensors for kinases and GTPases
Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226481/ https://www.ncbi.nlm.nih.gov/pubmed/21976697 http://dx.doi.org/10.1091/mbc.E11-01-0072 |
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author | Komatsu, Naoki Aoki, Kazuhiro Yamada, Masashi Yukinaga, Hiroko Fujita, Yoshihisa Kamioka, Yuji Matsuda, Michiyuki |
author_facet | Komatsu, Naoki Aoki, Kazuhiro Yamada, Masashi Yukinaga, Hiroko Fujita, Yoshihisa Kamioka, Yuji Matsuda, Michiyuki |
author_sort | Komatsu, Naoki |
collection | PubMed |
description | Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells. |
format | Online Article Text |
id | pubmed-3226481 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-32264812012-02-16 Development of an optimized backbone of FRET biosensors for kinases and GTPases Komatsu, Naoki Aoki, Kazuhiro Yamada, Masashi Yukinaga, Hiroko Fujita, Yoshihisa Kamioka, Yuji Matsuda, Michiyuki Mol Biol Cell Articles Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells. The American Society for Cell Biology 2011-12-01 /pmc/articles/PMC3226481/ /pubmed/21976697 http://dx.doi.org/10.1091/mbc.E11-01-0072 Text en © 2011 Komatsu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. |
spellingShingle | Articles Komatsu, Naoki Aoki, Kazuhiro Yamada, Masashi Yukinaga, Hiroko Fujita, Yoshihisa Kamioka, Yuji Matsuda, Michiyuki Development of an optimized backbone of FRET biosensors for kinases and GTPases |
title | Development of an optimized backbone of FRET biosensors for kinases and GTPases |
title_full | Development of an optimized backbone of FRET biosensors for kinases and GTPases |
title_fullStr | Development of an optimized backbone of FRET biosensors for kinases and GTPases |
title_full_unstemmed | Development of an optimized backbone of FRET biosensors for kinases and GTPases |
title_short | Development of an optimized backbone of FRET biosensors for kinases and GTPases |
title_sort | development of an optimized backbone of fret biosensors for kinases and gtpases |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226481/ https://www.ncbi.nlm.nih.gov/pubmed/21976697 http://dx.doi.org/10.1091/mbc.E11-01-0072 |
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