GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Förster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.
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July 2004
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Research Article|
June 22 2004
Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer
Satoshi KARASAWA;
Satoshi KARASAWA
*Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
†Amalgaam Co. Ltd, 2-9-3 Itabashi, Itabashi-ku, Tokyo, 173-0004, Japan
‡Medical & Biological Laboratories Co. Ltd, 3-5-10 Marunouchi, Naka-ku, Nagoya-city, 460-0002, Japan
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Toshio ARAKI;
Toshio ARAKI
*Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
†Amalgaam Co. Ltd, 2-9-3 Itabashi, Itabashi-ku, Tokyo, 173-0004, Japan
‡Medical & Biological Laboratories Co. Ltd, 3-5-10 Marunouchi, Naka-ku, Nagoya-city, 460-0002, Japan
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Takeharu NAGAI;
Takeharu NAGAI
*Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
§Structure and Function of Biomolecules, PRESTO, JST, 4-1-8 Hon-cho, Kawaguchi, Saitama, 332-0012, Japan
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Hideaki MIZUNO;
Hideaki MIZUNO
*Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
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Atsushi MIYAWAKI
Atsushi MIYAWAKI
1
*Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
1To whom correspondence should be addressed (e-mail matsushi@brain.riken.go.jp).
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Biochem J (2004) 381 (1): 307–312.
Article history
Received:
March 01 2004
Revision Received:
March 31 2004
Accepted:
April 05 2004
Accepted Manuscript online:
April 05 2004
Citation
Satoshi KARASAWA, Toshio ARAKI, Takeharu NAGAI, Hideaki MIZUNO, Atsushi MIYAWAKI; Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 1 July 2004; 381 (1): 307–312. doi: https://doi.org/10.1042/BJ20040321
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