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Comparative Study
. 2004 Jul 1;381(Pt 1):307-12.
doi: 10.1042/BJ20040321.

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

Satoshi Karasawa  1 , Toshio Araki, Takeharu Nagai, Hideaki Mizuno, Atsushi Miyawaki
Affiliations
Comparative Study

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

Satoshi Karasawa et al. Biochem J. .

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.

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Figures

Figure 1
Figure 1. Amino acid sequence (single-letter code) alignment of DsRed, mRFP1, KO, mKO and MiCy
In the sequence of MiCy, the β-sheet-forming regions are underlined. Residues whose side chains form the interior of the β-can [20] are shaded in grey. In the mRFP1 sequence, the substituted amino acids are shaded in yellow. In the sequence of mKO, the substituted amino acids that disrupt dimeric structure, increase folding efficiency and inhibit aggregation are shaded in blue, red and green respectively. In the sequences of both KO and mKO, the artificial regions are boxed in red. The residues responsible for chromophore synthesis are indicated by an asterisk.
Figure 2
Figure 2. Light-absorption properties of the new fluorescent proteins
Absorption spectra of MiCy (A), KO (B) and mKO (C), and the pH-dependence of the absorption (Abs.) peak at the maximum (D).
Figure 3
Figure 3. Normalized excitation (broken line) and emission (solid line) spectra of MiCy (A), KO (B) and mKO (C)
Fluo.Int., fluorescence intensity.
Figure 4
Figure 4. The equilibrium radial absorbance profiles at 25000 rev./min by analytical ultracentrifugation analysis for MiCy (59.7 kDa) (A), KO (77.0 kDa) (B), and mKO (28.1 kDa) (C)
Figure 5
Figure 5. FRET between MiCy and mKO
(A) Normalized excitation (broken line) and emission (solid line) spectra of MiCy and mKO. (B) Normalized excitation (broken line) and emission (solid line) spectra of CFP and YFP. (C) Primary structure of the caspase-3-sensor protein. (D) Emission spectra of the caspase-3-sensor with excitation at 440 nm before (light grey) and after (dark grey) incubation with caspase 3. Inset, overt appearance of the sample tubes. Fluo.Int., fluorescence intensity.
Figure 6
Figure 6. FRET imaging of caspase 3 activity during an apoptotic process
(A) FRET images of HeLa cells displayed in real colour and pseudo colour (ratios). Scale bar, 20 μM. (B) Time courses of the 559/495 nm emission ratios observed in the three cells indicated in (A).
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