A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.


Results
We aimed to develop a darker sREACh by error-prone PCR-based random mutagenesis. Mutations were introduced between amino acid positions Y143 and E235 ( Supplementary Fig. S1); we avoided introducing any mutations into the chromophore. The PCR products with random mutations were then fused to the rest of the sREACh sequence (corresponding to amino acid residues 1-142) and ligated into a bacterial expression vector, constructing a genetic library. To screen the library for a dark sREACh mutant that has a high extinction coefficient but a low quantum yield, we first identified vividly colored colonies under day light, assuming that this characteristic is due to high absorption by the fluorescent protein in the bacteria. After that, the identified colonies were examined under blue light: we searched for dark colonies using a method similar to the one described elsewhere 11 . During the screening, we found a bright yellow colony that showed no fluorescence under blue light. Sequence analysis revealed 4 mutations (V148H, Y203T, A206K, R223F) in sREACh ( Supplementary Fig. S1). These mutations are a combination of previously reported mutations 9,12,13 . The purified mutant has a yellow color under daylight and shows greatly reduced fluorescence under blue light (Fig. 1a). Spectral analysis confirmed an absorption peak at 486 nm and an emission peak at 510 nm ( Fig. 1b-d and Table 1), similar to those of mEGFP. This blue-shifted spectrum of the sREACh mutant is most likely due to the Y203T mutation, which disrupts the π -stack 12 . Because this new mutant has very weak fluorescence and spectral properties similar to those of EGFP, we named this protein ShadowG where G stands for "green". Further analysis revealed that the molar extinction coefficient of ShadowG is 89,000 M −1 ·cm −1 , comparable to that of sREACh (Table 1). Quantum efficiency of ShadowG is 0.005, which is 14-fold smaller than that of sREACh (0.07; Table 1). Consistent with these results, the fluorescence lifetime of ShadowG (0.16 ns) is much shorter than that of sREACh (0.45 ns; Fig. 1e).
Next, to measure the folding kinetics, ShadowG was denatured in urea as described previously 14 , and the recovery of fluorescence was monitored after dilution in urea-free buffer. The fluorescence of denatured ShadowG recovered in 37s: faster than recovery of sREACh (308 seconds; Fig. 1f, Table 1). Next, chromophores of the urea-denatured ShadowG were reduced with dithionite, and reoxidation time and recovery were monitored after dilution in urea-free buffer. Reoxidation time of ShadowG (76 min) is half that of sREACh (133 min; Fig. 1g, Table 1).
Because ShadowG is darker than sREACh in vitro, we next tested the bleed-through effect of ShadowG in live cells. Cells were cotransfected with a plasmid encoding mCherry, ShadowG, or sREACh and a plasmid encoding mEGFP fused to the CAAX motif of K-Ras (Fig. 1h), and the fluorescence and its lifetime were monitored in the green channel with the band pass filter (letting wavelengths 470-550 nm through) under a 2-photon fluorescence lifetime imaging microscope (2pFLIM). Although mEGFP-CAAX was localized to the plasma membrane, mCherry, ShadowG, and sREACh were localized to the cytosol; therefore, the shorter lifetime of fluorescence of the cytosol was due to spectral contamination with these fluorescent proteins. Notably, when mEGFP-CAAX was coexpressed with sREACh, weak fluorescence with short lifetime was observed in the cytosol (Fig. 1h). This cytosolic fluorescence was removed by means of a narrow band pass filter (letting wavelengths 500-520 nm through), which blocks most of YFP fluorescence and a half of GFP fluorescence. This result suggests that cytosolic fluorescence with short lifetime was due to the spectral contamination with sREACh. The significant fluorescence contamination is not seen with mCherry and ShadowG. Quantification of fluorescence lifetime further confirmed that ShadowG yields significantly lesser fluorescence contamination (Fig. 1i).
Next, we characterized maturation efficiency and FRET efficiency of ShadowG when it was paired with mEGFP in live HeLa cells as described previously 10 . We expressed a tandem construct consisting of mEGFP and ShadowG, and the fluorescence lifetime of mEGFP was measured under the 2pFLIM (Fig. 2a-c). Since the fluorescence lifetime decay curves are convolution of both the FRET efficiency and maturity of acceptor, we measured these parameters separately for more detailed information, as described previously (Fig. 2d,e) 10,15 . The FRET efficiency of mEGFP-ShadowG was better than that of mEGFP-mCherry and mEGFP-sREACh, suggesting that mEGFP and ShadowG are a good FRET pair (Fig. 2d). When the maturation efficiency of ShadowG was compared with those of mCherry and sREACh, ShadowG showed higher maturation efficiency than mCherry but lower maturation efficiency in comparison with sREACh (Fig. 2e). Although, maturation efficiency is estimated by the fluorescence lifetime curve fitting using tandem fluorescent protein, there is a possibility that inter-molecular FRET between Scientific RepoRts | 5:15334 | DOi: 10.1038/srep15334 tandem fluorescent proteins due to the dimerization could affect this measurement, since fluorescent proteins tend to form dimer 13 . Therefore, to exclude this possibility, we measured the inter-molecular FRET between tandem fluorescent proteins, using Y66L chromophore-killed mutant 16 , and found that no significant dimer fraction exists in our experimental condition (Fig. 2f).
To further test the performance of ShadowG, we compared the three single-molecule types of the CaMKII FRET sensor (Camuiα ) design differing in their acceptor fluorophores (Fig. 3). mEGFP was  fused to the N terminus, and mCherry, sREACh, and ShadowG were fused to the C terminus of full-length CaMKIIα : a configuration similar to the one previously reported [17][18][19] . The binding of Ca 2+ /calmodulin induced a structural change of CaMKII, leading to activation of CaMKIIα and a decrease in FRET (Fig. 3a). We transfected HeLa cells with one of the three CaMKII sensors and compared the response signals under the 2pFLIM 7,18-20 . After stimulation with a 10 μ M ionophore, CaMKII was rapidly activated, and the signal reached a plateau within a few minutes ( Fig. 3b-g). When the FRET response signal of mEGFP-CaMKII-ShadowG was compared with that of mCherry and sREACh, the signal was similar to that of mCherry and weaker than that of sREACh ( Fig. 3d). Nonetheless, the response variability of individual cells was advantageously reduced with mEGFP-CaMKII-ShadowG than with either the mCherry or sREACh version ( Fig. 3h-j). These data indicated that ShadowG-based Camuiα produces a stable response superior to that of the mCherry and sREACh versions. Next, we compared the three single-molecule types of LOV2 FRET constructs differing in their acceptor fluorophores (Fig. 4). The structure of the LOV2-Jα helix domain (which is a light-sensitive domain of Phototropin 1) is changed by absorption of blue light 21 . Namely, in the absence of blue light, it assumes a closed conformation, but after absorption of blue light, this domain reversibly opens 22 (Fig. 4a). To evaluate the performance of ShadowG, we monitored FRET signals after the light-dependent structural changes in LOV2. mEGFP was fused to the N terminus of LOV2-Jα helix, and mCherry, sREACh, or ShadowG were fused to the C terminus, respectively. We transfected HeLa cells with one of the three LOV2 constructs and monitored the blue-light-dependent structural changes in the LOV2 FRET probes under the 2pFLIM ( Fig. 4a-g). Decay was 40 s, consistent with another study 22 . When the FRET response signal of mEGFP-LOV2-ShadowG was compared with that of the mCherry and sREACh versions, the former signal was stronger than that of the mCherry construct and similar to that of the sREACh construct (Fig. 4d). As with Camuiα , the response variability of individual cells was reduced more strongly with mEGFP-LOV2-ShadowG than with the mCherry and sREACh versions ( Fig. 4h-j). These data indicates that ShadowG-based LOV2 produces a stable response superior to that of the mCherry and sREACh versions. Furthermore, the reduced variability of ShadowG in comparison with sREACh was confirmed in HEK293 cells and dissociated neurons ( Supplementary Fig. S2, S3).
Next, ShadowG was applied to the previously reported separate-type H-Ras FRET sensor (FRas2-M) 23 , and was compared the FRET signal with that of mCherry (Fig. 5). We only compared with mCherry, but not with sREACh, since it has bleed-thorough effect (Fig. 1h, i). We used mEGFP-H-Ras and the Ras-binding domain (RBD)-ShadowG via the P2A sequence to ensure equal expression of these molecules 24 and to minimize the response variability due to the imbalanced expression of the donor and acceptor. We transfected HeLa cells with this modified FRas2-M and compared the response signals under the 2pFLIM. After stimulation with epidermal growth factor (EGF), H-Ras was rapidly activated (within a few minutes; Fig. 5b,c,e,f). When the FRET response signal of Ras sensor with ShadowG was compared with that of mCherry, the former signal was stronger (Fig. 5d), suggesting that ShadowG is superior to mCherry as an acceptor. Reduced variability of basal FRET and the response signal-which was seen in the CaMKII and LOV2 sensor-was not observed (Fig. 5g, h).
By taking advantage of the narrow bandwidth of FRET in the pair mEGFP-ShadowG, we simultaneously imaged H-Ras activation and translocation of ERK into the nucleus after stimulation with EGF (Fig. 6). ERK is a well-known kinase which is phosphorylated by the Ras/Raf/MEK signaling cascade 25 . After phosphorylation, ERK translocates into the nucleus where it phosphorylates transcription factors. HeLa cells were transfected with Ras sensor, mCherry-ERK2, and MEK1 for retention of ERK2 in the nucleus 26,27 , and Ras activation and mCherry were monitored under the 2pFLIM and by means of 2-photon fluorescence microscopy, respectively (Fig. 6a). After stimulation with EGF, Ras was rapidly activated, and the signal decayed in 25 min (Fig. 6b). Delayed by the Ras activation, ERK2 showed slow translocation into the nucleus (Fig. 6c). These observations demonstrated that the mEGFP-ShadowG FRET pair enables the dual observation with mCherry.

Discussion
In this work, we successfully developed a new fluorescent protein, ShadowG, as a FLIM-FRET acceptor for pairing with mEGFP. ShadowG is darker than the previously reported dark YFP mutants, such as sREACh 10 . The new protein reduces the risk of artifacts due to spectral contamination ( Fig. 1) and has folding and maturation kinetics better than those of previously reported dark fluorescent proteins such as sREACh and Ultramarine 10,28 . Another advantage of ShadowG is its signal stability. When ShadowG was applied to a CaMKII or LOV2 FRET sensor, HeLa cells expressing these sensors showed smaller basal FRET variability in comparison with mCherry and sREACh constructs (Figs 3 and 4). Similar results were obtained with HEK293 and hippocampal neurons ( Supplementary Fig. S2, S3). One simple explanation of the minimized variability is that sensor quality is improved due to the superior protein folding (Fig. 1f, g) 19 . Although maturation efficiency of ShadowG in tandem construct is smaller than that of sREACh (Fig. 2), the response signal of the ShadowG FRET sensor is comparable to that of the sREACh, depending on the sensor type (Fig. 4).
The contamination of the donor channel with acceptor fluorescence interferes with the acquisition of a correct FRET signal, especially with a hetero-FRET sensor that shows differential localization within the cell. However, since ShadowG has very low quantum efficiency, it could be applied to a hetero-type FRET sensor (Fig. 5) and used for dual observation with another signaling molecule. Taken together, these results show that ShadowG is a promising acceptor for precise multi-color FLIM-FRET.    (h-j), respectively. The data are also presented as mean ± SD on the right. Asterisks denote statistical significance (p < 0.05, t test). Plasmid construction. For construction of the EGFP-CAAX vector (used in Fig. 1h), the DNA sequence of the CAAX motif of K-Ras (corresponding to amino acid residues 173-188) was inserted into pEGFP-C1 (Clontech) with a linker encoding the peptide SGLRSRAQASNSAV. To create the cytosolic mCherry, sREACh, or ShadowG (used in Fig. 1h), we inserted the respective genes into the modified pEGFP-C1 vector, replacing EGFP. To create the tandem fluorescent protein constructs (used in Fig. 2), mCherry, sREACh, or ShadowG were inserted into multiple cloning site in the modified pEGFP-C1 vector, respectively. For construction of mEGFP-CaMKIIα -mCherry, mEGFP-CaMKIIα -sREACh, or mEGFP-CaMKIIα -ShadowG, mEGFP was fused to the N terminus of the full-length CaMKIIα sequence    (h-j), respectively. The data are also presented as mean ± SD on the right. Asterisks denote statistical significance (p < 0.05, t test).   with the linker peptide SGLRSRA. Subsequently, mCherry, sREACh, or ShadowG was subcloned into the C-terminal region of CaMKIIα with a linker encoding the peptide GSNQQIFLRDIEQVPQQ. For construction of the mEGFP-LOV2-mCherry, mEGFP-LOV2-sREACh, or mEGFP-LOV2-ShadowG vector, we fused mEGFP (DNA sequence corresponding to amino acid residues 1-235) to the N terminus of LOV2 domain (DNA sequence corresponding to amino acid residues 404-546 in Phototropin1) with a linker encoding the peptide ASM. Then, mCherry, sREACh, or ShadowG was subcloned into the C-terminal region of LOV2 with the linker peptide KLGNS. For construction of the H-Ras FRET sensors, we fused mCherry, sREACh, or ShadowG to the RBD of Raf1 (amino acid residues 50-131) with F-Ras2-M mutations (K65E, K108A) 23 and the linker peptide GSG. Subsequently, mEGFP-Ras with the linker peptide SGLRSRG was fused to the C terminus of the above protein via the P2A sequence 24 so that the RBD and H-Ras parts were translated into different polypeptides within the cell. For construction of mCherry-ERK2 and MEK1 plasmids, the source pCMV5-ratERK2-WT and EYFP-ERK2 plasmids were provided by Natalie Ahn (Addgene plasmid # 40812) 29 and by Akihiko Yoshimura, respectively. mCherry and ERK2 or MEK1 was subcloned into the modified pmEGFP-C1, replacing mEGFP.
Fluorescence properties of the fluorescent proteins. For bacterial expression of the His-tagged mEGFP, sREACh, or ShadowG, the respective genes were inserted into the pRSET vector (Invitrogen). His-tagged fluorescent proteins were overexpressed in Escherichia coli DH5α cells and purified on a Ni + -nitrilotriacetate column (HiTrap, GE Healthcare). Absorption and emission spectra of the fluorescent proteins that were diluted in PBS were measured on a UV-Vis spectrophotometer (UV-1800; Shimadzu) or a fluorescence spectrophotometer (RF-5300PC; Shimadzu), respectively. The molar concentration of the purified proteins was measured by the Bradford protein assay (Bio-Rad), considering the molecular weights predicted for the His-tagged fluorescent proteins. The molar extinction coefficients were determined by dividing the optical density of proteins by the molar concentration. The quantum efficiency (QY) of proteins was determined by comparison with QY (0.925) of fluorescein diluted in 0.1 M NaOH, as described previously 30 . Refolding and reoxidation kinetics. To analyze the refolding kinetics of sREACh and ShadowG, we denatured the proteins and refolded them as described previously 14 . Briefly, the purified proteins were dissolved in denaturation buffer (8 M urea, 1 mM dithiothreitol) and heated at 95 °C for 5 min for denaturation. We initiated the refolding reactions by diluting the denatured protein with a 100-fold volume of renaturation buffer (5 mM KCl, 2 mM MgCl 2 , 50 mM Tris pH 7.5, 1 mM dithiothreitol) at room temperature. In the reoxidation experiment, 5 mM dithionite was added to the denaturation buffer to reduce the chromophore. The fluorescence recovery was measured using a fluorescence spectrophotometer at 510 nm (RF-5300PC; Shimadzu).

Dissociated culture of hippocampal neurons and transfection. Cultured hippocampal neurons
were prepared as described elsewhere 31 . Briefly, postnatal day 1 rats were anesthetized and decapitated, followed by brain removal and hippocampal tissue dissection. Neurons triturated by papain treatment were plated at a density of 1.3 × 10 4 cells/cm 2 on polyethyleneimine-coated 35 mm culture dishes and maintained in serum-free Neurobasal medium supplemented with 2% B27, 2 mM GlutaMAX-I, and 10 mM HEPES at 37 °C and 5% CO 2 . After 11 days, neurons were transfected with 2 μ g of either mEGFP-LOV2-sREACh or mEGFP-LOV2-ShadowG plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacture's protocol. After 24-48 hours, the neurons in the buffer (10 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and Tris-base for adjusting to pH 7.4) were imaged. Two-photon fluorescence lifetime imaging. Details of the 2pFLIM-FRET imaging were described previously 7 . Briefly, mEGFP in the FRET sensor was excited with a Ti-sapphire laser (Mai Tai; Spectra-Physics) tuned to 920 nm. The scanning mirror (6210H; Cambridge Technology) was controlled with a PCI board (PCI-6110; National Instruments) and ScanImage software 32 . The green fluorescence photon signals were collected by an objective lens (60× , 0.9 NA; Olympus) and a photomultiplier tube (H7422-40p; Hamamatsu) placed after a dichroic mirror (565DCLP; Chroma) and emission filter (FF01-510/84 or FF03-510/20 in Fig. 1h; Semrock). For dual observation of H-Ras activation and mCherry-ERK2 translocation, a Ti-sapphire laser tuned to 980 nm was used for simultaneous excitation of mEGFP and mCherry. The green fluorescence was acquired as described above, and mCherry's fluorescent signal was Scientific RepoRts | 5:15334 | DOi: 10.1038/srep15334 collected by a photomultiplier tube (R3896; Hamamatsu) placed after the emission filter (FF01-625/90; Semrock). Measurement of fluorescence lifetime was carried out using a time-correlated single-photon counting board (SPC-150; Becker & Hickl) controlled with custom software 7 . For construction of a fluorescence lifetime image, the mean fluorescence lifetime in each pixel was translated into a color-coded image 33 . Analysis of the lifetime changes in individual cells was conducted as described previously 18 . Quantification of the FRET efficiency and mature fraction. To measure these parameters of the acceptor fluorescent protein in the tandem fluorescent protein in HeLa cells, we fitted the fluorescence lifetime curve to a double exponential function convolved with an instrument response function, G(t) 7 , assuming that two fractions exist in the cells: 1) mature mEGFP fused to an immature acceptor fluorescent protein where fluorescence lifetime of mEGFP (τ mEGFP ) is 2.59 ns; 2) mature mEGFP fused to a mature acceptor fluorescent protein where FRET occurs and fluorescence lifetime of mEGFP (τ FRET ) gets shorter: where P 1 and P 2 are the populations of mEGFP fused to the mature acceptor and immature acceptor, respectively. The mean FRET efficiency (Y FRET ) between mEGFP and the mature acceptor was calculated as follows: FRET FRET mEGFP and the fraction of the mature acceptor (A mature ) was calculated using the following formula: