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

• Karsten Weis

  editor

Bibliographic Information

Other Title

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

Abstract

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.

Journal

• Molecular biology of the cell

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

  American Society for Cell Biology

Keywords

• Cell Biology
• Molecular Biology

Details

• CRID: 1050845760653835776
• NII Article ID: 120003644367
• NII Book ID: AA10830484
• ISSN: 10591524
• ISSN: 19394586
• DOI: 10.1091/mbc.e11-01-0072
• HANDLE: 2433/151847
• Text Lang: en
• Article Type: journal article
• Data Source:
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  • Crossref
  • CiNii Articles
  • KAKEN