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Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer.
Biochem J. 2004 Jul 01; 381(Pt 1):307-12.BJ

Abstract

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.

Authors+Show Affiliations

Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15065984

Citation

Karasawa, Satoshi, et al. "Cyan-emitting and Orange-emitting Fluorescent Proteins as a Donor/acceptor Pair for Fluorescence Resonance Energy Transfer." The Biochemical Journal, vol. 381, no. Pt 1, 2004, pp. 307-12.
Karasawa S, Araki T, Nagai T, et al. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J. 2004;381(Pt 1):307-12.
Karasawa, S., Araki, T., Nagai, T., Mizuno, H., & Miyawaki, A. (2004). Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. The Biochemical Journal, 381(Pt 1), 307-12.
Karasawa S, et al. Cyan-emitting and Orange-emitting Fluorescent Proteins as a Donor/acceptor Pair for Fluorescence Resonance Energy Transfer. Biochem J. 2004 Jul 1;381(Pt 1):307-12. PubMed PMID: 15065984.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. AU - Karasawa,Satoshi, AU - Araki,Toshio, AU - Nagai,Takeharu, AU - Mizuno,Hideaki, AU - Miyawaki,Atsushi, PY - 2004/04/05/accepted PY - 2004/03/31/revised PY - 2004/03/01/received PY - 2004/4/7/pubmed PY - 2004/11/5/medline PY - 2004/4/7/entrez SP - 307 EP - 12 JF - The Biochemical journal JO - Biochem. J. VL - 381 IS - Pt 1 N2 - 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. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/15065984/Cyan_emitting_and_orange_emitting_fluorescent_proteins_as_a_donor/acceptor_pair_for_fluorescence_resonance_energy_transfer_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20040321 DB - PRIME DP - Unbound Medicine ER -