2003 Fiscal Year Final Research Report Summary
Development of sensor peptide based on fluorescence resonance energy transfer
| Project/Area Number |
13650852
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | National Institute of Advanced Industrial Science and Technology (AIST) (2002-2003)
Japan Advanced Institute of Science and Technology (2001) |
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Principal Investigator |
YOKOYAMA Kenji National Institute of Advanced Industrial Science and Technology (AIST), Research Center of Advanced Bionics, Deputy Director, バイオニクス研究センター, 副センター長 (80242121)
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| Project Period (FY) |
2001 – 2003
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| Keywords | protein kinase A / fluorescence resonance energy transfer / phosphorylation / solid phase synthesis / fluorophore / quencher |
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| Research Abstract |
We are currently focusing on a peptide as a novel biosensor material and have investigated several sensor peptides that can be applied to analyzing intracellular signal transduction. We have designed a fluorescence resonance energy transfer based sensor peptide for analyzing protein kinase activity. For instance, we have synthesized a sensor peptide modified with EDANS and dabcyl for monitoring MAPKK activity. In this work, we synthesized 10mer and 15mer peptides, which include amino acid sequence of phosphorylated site, ArgArgAlaSerLeu, of c-AMP dependent protein kinase (PKA). Ser is phosphorylated by PKA in the cell. EDANS-modified 10mer and 15mer peptides, GLRRASLGE(EDANS)G, and GDENLRRASLGEDE(EDANS)G. were synthesized on bead. After cleavage and deprotection of peptide side chain, dabcyl group was attached at the N-terminal O-amino group. Ser-phosphorylated sensor peptides were also synthesized.
Fluorescence property of the peptides modified with EDANS and dabcyl was investigated. Energy transfer from EDANS to dabcyl, quenching by dabcyl, was reduced by phosphorylation of Ser. Peptide main chain was stretched by increase in hydrophilicity, and the distance between the fluorescence dye and quencher increased.
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