Development of an optimized backbone of FRET biosensors for kinases and GTPase

• Komatsu, Naoki

• 青木, 一洋

• Yamada, Masashi

• Yukinaga, Hiroko

• Fujita, Yoshihisa

• Kamioka, Yuji

• Matsuda, Michiyuki

• Karsten Weis

  editor

書誌事項

タイトル別名

• Development of an optimized backbone of FRET biosensors for kinases and GTPases.

抄録

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.

収録刊行物

• Molecular biology of the cell

  Molecular biology of the cell 22 (23), 4647-4656, 2011-12

  American Society for Cell Biology

キーワード

• Cell Biology
• Molecular Biology

詳細情報

• CRID: 1050845760653835776
• NII論文ID: 120003644367
• NII書誌ID: AA10830484
• ISSN: 10591524
• ISSN: 19394586
• DOI: 10.1091/mbc.e11-01-0072
• HANDLE: 2433/151847
• 本文言語コード: en
• 資料種別: journal article
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