A single circularly permuted GFP sensor for inositol-1,3,4,5-tetrakisphosphate based on a split PH domain |
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| Authors: | Reiko Sakaguchi Takashi Endoh Seigo Yamamoto Kazuki Tainaka Kenji Sugimoto Nobutaka Fujieda Shigeki Kiyonaka Yasuo Mori Takashi Morii |
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| Institution: | 1. Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan;2. CREST, JST, Uji, Kyoto 611-0011, Japan;3. Pioneering Research Unit for Next Generation, Kyoto University, Uji, Kyoto 611-0011, Japan;4. Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan;5. CREST, JST, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan |
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| Abstract: | A fluorescent sensor for the detection of inositol-1,3,4,5-tetrakisphosphate, Ins(1,3,4,5)P4, was constructed from a split PH domain and a single circularly permuted GFP. A structure-based design was conducted to transduce a ligand-induced subtle structural perturbation of the split PH domain to an alteration in the population of the protonated and the deprotonated states of the GFP chromophore. Excitation of each distinct absorption band corresponding to the protonated or the deprotonated state of GFP resulted an increase and a decrease, respectively, in the intensity of emission spectra upon addition of Ins(1,3,4,5)P4 to the split PH domain-based sensor. The Ins(1,3,4,5)P4 sensor retained the ligand affinity and the selectivity of the parent PH domain, and realized the ratiometric fluorescence detection of Ins(1,3,4,5)P4. |
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