title: Development of an optimized backbone of FRET biosensors for kinases and GTPases.
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.
authors: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
authors: Naoki
authors: N
authors: Komatsu
authors: Kazuhiro
authors: K
authors: Aoki
authors: Masashi
authors: M
authors: Yamada
authors: Hiroko
authors: H
authors: Yukinaga
authors: Yoshihisa
authors: Y
authors: Fujita
authors: Yuji
authors: Y
authors: Kamioka
authors: Michiyuki
authors: M
authors: Matsuda
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000032:analysis*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000032:analysis*; D015202:Protein Engineering / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000032:analysis / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010770:Phosphotransferases; D020558:GTP Phosphohydrolases