New Donor−Acceptor Pair for Fluorescent Immunoassays by Energy Transfer

1999 ◽  
Vol 10 (6) ◽  
pp. 1107-1114 ◽  
Author(s):  
Uwe Schobel ◽  
Hans-Joachim Egelhaaf ◽  
Andreas Brecht ◽  
Dieter Oelkrug ◽  
Günter Gauglitz
Keyword(s):  
Energy Transfer ◽  
Acceptor Pair ◽  
Donor Acceptor ◽  
Donor Acceptor Pair

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.

scholarly journals Highly efficient fluorescence resonance energy transfer in co-encapsulated BODIPY nanoparticles

2021 ◽  
Vol 12 (4) ◽  
pp. 361-367
Author(s):  
Priyadarshine Hewavitharanage ◽  
Launa Steele ◽  
Isaac Dickenson
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
Highly Efficient ◽  
Fluorescence Measurements ◽  
Donor Acceptor ◽  
Donor Acceptor Pair ◽  
Fluorescence Resonance

Fluorescence resonance energy transfer (FRET) is a powerful tool used in a wide range of applications due to its high sensitivity and many other advantages. Co-encapsulation of a donor and an acceptor in nanoparticles is a useful strategy to bring the donor-acceptor pair in proximity for FRET. A highly efficient FRET system based on BODIPY-BODIPY (BODIPY:  boron-dipyrromethene) donor-acceptor pair in nanoparticles was synthesized. Nanoparticles were formed by co-encapsulating a green emitting BODIPY derivative (FRET donor, lmax = 501 nm) and a red emitting BODIPY derivative (FRET acceptor, lmax = 601 nm) in an amphiphilic polymer using the precipitation method. Fluorescence measurements of encapsulated BODIPY in water following 501 nm excitation caused a 3.6 fold enhancement of the acceptor BODIPY emission at 601 nm indicating efficient energy transfer between the green emitting donor BODIPY and the red emitting BODIPY acceptor with a 100 nm Stokes shift. The calculated FRET efficiency was 96.5%. Encapsulated BODIPY derivatives were highly stable under our experimental conditions.

scholarly journals Homologous versus heterologous interactions in the bicomponent staphylococcal γ-haemolysin pore1

2006 ◽  
Vol 394 (1) ◽  
pp. 217-225 ◽  
Author(s):  
Gabriella Viero ◽  
Romina Cunaccia ◽  
Gilles Prévost ◽  
Sandra Werner ◽  
Henri Monteil ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Pore Formation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Wild Type ◽  
Acceptor Pair ◽  
Donor Acceptor ◽  
Left And Right ◽  
Donor Acceptor Pair ◽  
The Right

Staphylococcal γ-haemolysin HlgA–HlgB forms a β-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues – placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor–acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA–HlgB or HlgB–HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA–HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB–HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.

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