scholarly journals Spectral Profiling for the Simultaneous Observation of Four Distinct Fluorescent Proteins and Detection of Protein-Protein Interaction via Fluorescence Resonance Energy Transfer in Tobacco Leaf Nuclei

2002 ◽  
Vol 129 (3) ◽  
pp. 931-942 ◽  
Author(s):  
Naohiro Kato ◽  
Dominique Pontier ◽  
Eric Lam
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interaction ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Simultaneous Observation ◽  
Tobacco Leaf ◽  
Protein Protein Interaction ◽  
Fluorescence Resonance

scholarly journals Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer

2004 ◽  
Vol 381 (1) ◽  
pp. 307-312 ◽  
Author(s):  
Satoshi KARASAWA ◽  
Toshio ARAKI ◽  
Takeharu NAGAI ◽  
Hideaki MIZUNO ◽  
Atsushi MIYAWAKI
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
Donor And Acceptor ◽  
Donor Acceptor ◽  
Donor Acceptor Pair ◽  
Fluorescence Resonance

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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