A new bright green-emitting fluorescent protein – engineered monomeric and dimeric forms Robielyn P. Ilagan1, Elizabeth Rhoades1, David F. Gruber2, Hung-Teh Kao3, Vincent A. Pieribone4 and Lynne Regan1,5

1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA 2 Department of Natural Sciences, and The Graduate Center, City University of New York, NY, USA 3 Department of Psychiatry and Human Behavior, , Providence, RI, USA 4 The John B. Pierce Laboratory, Yale University, New Haven, CT, USA 5 Department of Chemistry, Yale University, New Haven, CT, USA

Keywords Fluorescent proteins have become essential tools in molecular and biologi- detection marker; fluorescence correlation cal applications. Here, we present a novel fluorescent protein isolated from spectroscopy; fluorescent protein; warm water coral, Cyphastrea microphthalma. The protein, which we oligomeric states; relative brightness named vivid Verde fluorescent protein (VFP), matures readily at 37 °C and Correspondence emits bright green light. Further characterizations revealed that VFP has a L. Regan Department of Molecular tendency to form dimers. By creating a homology model of VFP, based on Biophysics and Biochemistry, Yale the structure of the red fluorescent protein, DsRed, we were able to make University, New Haven, CT 06520, USA mutations that alter the protein’s oligomerization state. We present two Fax: (203) 432 5175 proteins, mVFP and mVFP1, that are both exclusively monomeric, and Tel: (203) 432 9843 one protein, dVFP, which is dimeric. We characterized the spectroscopic E-mail: [email protected] properties of VFP and its variants in comparison with enhanced green fluo- Note rescent protein (EGFP), a widely used variant of GFP. All the VFP vari- The nucleotide sequence data are available ants are at least twice as bright as EGFP. Finally, we demonstrated the in the DDBJ ⁄ EMBL ⁄ GenBank databases effectiveness of the VFP variants in both in vitro and in vivo detection under the accession number FN597286 and applications. the protein sequence data are in Uni- ProtKB ⁄ TrEMBL with the accession number Structured digital abstract D1J6P8. l MINT-7709188: VFP (uniprotkb:D1J6P8) and VFP (uniprotkb:D1J6P8) bind (MI:0407) by classical fluorescence spectroscopy (MI:0017) (Received 22 December 2009, revised 5 l MINT-7709201: VFP (uniprotkb:D1J6P8) and VFP (uniprotkb:D1J6P8) bind (MI:0407) by February 2010, accepted 15 February 2010) fluorescence correlation spectroscopy (MI:0052) l MINT-7709216, MINT-7709247, MINT-7709237: VFP (uniprotkb:D1J6P8) and VFP (uni- doi:10.1111/j.1742-4658.2010.07618.x protkb:D1J6P8) bind (MI:0407) by molecular sieving (MI:0071)

Introduction Fluorescent proteins (FPs) have become ubiquitous researchers have sought new variants of this protein, tools in biological and biomedical research. Since the as well as of other FPs, with properties that are cloning and exogenous expression of green fluorescent well-suited for a particular application [1–3]. Extensive protein (GFP) from the jellyfish Aequorea victoria, mutagenesis has been performed on FPs to better

Abbreviations dVFP, dimeric VFP; EC, extinction coefficient; EGFP, enhanced GFP; FCS, fluorescence correlation spectroscopy; FMRP, fragile X mental retardation protein; FP, fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; hpf, hours post-fertilization; mVFP, monomeric VFP; QY, quantum yield; tD, time; t½, half time; T-Mod, TPR-based recognition module; TPR, tetratricopeptide repeats; VFP, vivid Verde fluorescent protein.

FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS 1967 Monomeric and dimeric forms of green-emitting FP R. P. Ilagan et al. tailor their properties to the needs of biologists these FPs display a higher degree of oligomerization, [1,2,4,5]. Of special interest are FPs with new excita- which is detrimental for cellular labeling [17,18,23]. To tion and emission wavelengths, FPs with increased overcome FP oligomerization, mutations must be made brightness, FPs that are monomeric and FPs that at the monomer–monomer interface. The exact nature mature rapidly at 37 °C. of such interfaces varies depending on the nature and GFP is a 238-amino-acid protein, whose chromo- origin of the FP [2]. phore is formed by the post-translational re-arrange- Many FPs, either isolated from natural sources or ment of an internal Ser-Tyr-Gly sequence to a engineered from GFP or DsRed, are known and avail- 4-(p-hydroxybenzylidene)-imidazolidine-5-one structure able [1,2]. However, only a few of the FPs are [6]. The crystal structure of GFP revealed that the widely used in various cell-imaging applications and chromophore is buried in the center of a b-barrel most of them have certain limitations [1,2,24]. structure [7,8]. Amino acid mutagenesis and protein A continuing effort must be made to improve the engineering were carried out on GFP to improve its spectral characteristics and stabilities of the FPs, or spectral characteristics, oligomeric state and chromo- alternatively, to search for new FPs with optimal phore-maturation at 37 °C [6,9–12]. A broad range of properties, for maximum utility in cellular imaging. GFP variants with fluorescence emission ranging from The natural habitat of A. victoria is the cool waters blue to yellow regions of the visible spectrum was cre- off the northwest coast of Washington State. One ated [1,2]. Enhanced green fluorescent protein (EGFP) might expect organisms that inhabit warmer waters to is a widely used variant of GFP, which has mutations have evolved FPs that mature more rapidly at higher at two positions: F64L and S65T [9,10]. EGFP is . Here we describe the characterization brighter and matures more rapidly at 37 °C than wild- and modification of a novel FP that was isolated from type GFP [1,9]. Protein engineering of EGFP has Cyphastrea microphthalma, a scleractinian coral found yielded several green variants with improved character- in the warmer waters of the Australian Great Barrier istics, such as Emerald FP. This Emerald FP has (Fig. 1). Several new fluorescent organisms were improved photostability and brightness compared with identified by diving at night with UV illumination, and EGFP [11]. Another GFP variant is the ‘superfolder’ the FPs were cloned from these organisms and GFP that is designed to fold faster at 37 °C. This ‘su- expressed in Escherichia coli [25,26]. We found that the perfolder’ GFP is also brighter and more acid resistant vast majority of proteins characterized indeed mature than either EGFP or Emerald FP [12]. A weak ten- robustly and rapidly at 37 °C. Here we report the dency of GFP and its variants to dimerize was com- properties of one of the novel green-emitting FPs, pletely eliminated using point mutations at F223K, vivid Verde FP (VFP), which exhibits useful proper- L221K, or A206K [13,14]. ties. VFP is very bright, matures rapidly at 37 °C and Another FP, DsRed, from the sea anemone Disco- we have engineered exclusively monomeric or dimeric soma striata, is also of great interest to researchers variants of it. These properties are particularly well because its intrinsic fluorescence is red rather than green suited to a variety of molecular and biological applica- [15,16]. The chromophore of DsRed is closely related to tions. that of GFP, being formed by the re-arrangement of an internal Gln-Tyr-Gly tripeptide [15]. The extended Results conjugation in the chromophore causes the red-shift observed in DsRed and other red FPs [4]. DsRed forms Sequence of the new FP and relation to other a strong tetramer both in and in crystal and its known FPs chromophore maturation is very slow [17–19]. As a result of these limitations, DsRed has been a target of A new FP, VFP, was isolated and cloned from the protein engineering and mutations to improve its C. microphthalma coral, collected in 1.2 m of water off chromophore maturation rate and to reduce oligomeri- Lizard Island on the Australian zation [20–22]. A directed evolution approach was [25,26]. The alignment of the amino acid sequences of performed on DsRed to make a monomeric version, VFP, DsRed and EGFP is shown in Fig. 1A. The mRFP1, which has a total of 33 amino acid mutations amino acid residues that form the chromophore are in [21]. In addition to DsRed, there are many other FPs, bold and underlined. The chromophore residues at ranging from blue-, cyan-, green- and yellow- to positions 66, 67 and 68, following the amino acid resi- red-emitting, which have different spectral properties, dues numbering in DsRed, are QYG in VFP, QYG in brightness, and stabilities, that have been isolated from DsRed and TYG in EGFP. VFP shows greater reef corals and other Anthozoa species [1,2]. Most of sequence identity overall to DsRed than to EGFP,

1968 FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS R. P. Ilagan et al. Monomeric and dimeric forms of green-emitting FP

A

Fig. 1. (A) Amino acid sequence alignment of VFP with DsRed and EGFP. The chromo- phore-forming amino acid residues are shown in bold and are underlined. The amino acid residues (N158 and T160) of VFP, where the mutations were made, are indicated by a bold letter. The conserved Arg and Glu (corresponding to Arg96 and Glu222 of GFP) residues are shown on a BC gray background. (B) A scleractinian coral, Cyphastrea microphthalma, collected in 1.2 m of water off Lizard Island on the Aus- tralian Great Barrier Reef. (C) Overlay of the absorption (abs), fluorescence-excitation (ex) and fluorescence-emission (em) spectra of VFP. The samples were excited at 450 nm and the emission spectra were measured from 465 to 650 nm. The fluorescence exci- tation spectra were obtained from 250 to 515 nm by monitoring the emission at 530 nm. The spectra were normalized at the maximum peak.

with 53% sequence identity to DsRed and only 20% at 475 and 499 nm, respectively [17]. However, it has sequence identity to EGFP. Sequence alignment dem- also been proposed that the red-emitting chromophore onstrates the conservation of many positions in VFP, of DsRed and of related chromoproteins is produced which are presumably structurally and ⁄ or functionally from a blue-emitting neutral form of a GFP-like important. Arg96 and Glu222 of GFP, which were chromophore, the green anionic species being the proposed to participate in chromophore maturation dead-end product [32]. The GFP-like chromophore [27], are also conserved in DsRed and VFP. The VFP of VFP is stable and further conversion into the coding sequence was deposited in the EMBL nucleo- red-emitting chromophore was not observed. sequence database under the accession number Two tryptophan residues at positions 93 and 143 of FN597286. Using the Swiss Institute of Bioinformatics DsRed, located in the immediate vicinity of the chro- BLAST Network Service, the VFP sequence was found mophore, are conserved in VFP (corresponding to to have the highest sequence identity, of 83%, to a positions 89 and 139). Thus, the absorption spectrum GFP isolated from coral Montastraea cavernosa [28]. of VFP showed a peak at 280 nm (Fig. 1C) as a result Sequence alignment also showed that there are several of the presence of these Trp residues, and excitation at cyan, green, or red FPs and chromoproteins from 280 nm gave an emission peak at 503 nm. coral in which the chromophore is formed by amino acids QYG, the same as in VFP. Oligomeric state of VFP VFP exhibits maximum excitation and emission peaks at 491 and 503 nm, respectively, as shown in For many applications, it is essential that the FP used Fig. 1C. These spectral properties are more similar to to ‘tag’ another protein is monomeric [24]. If an FP is those of EGFP rather than to those of DsRed, despite not monomeric, then its oligomerization may influence the fact that the sequence of VFP is more closely the behavior of the tagged protein, thus perturbing the related to DsRed than to EGFP. The chromophore system under study. We used gel-filtration chromatog- formation in GFP involves cyclization, oxidation and raphy to assess the oligomeric state of VFP. To allow dehydration, and in DsRed and other coral FPs, an direct comparison with a known protein, we also puri- additional oxidation step occurs [4,29–31]. Previously, fied EGFP, which is monomeric at DsRed chromophore maturation has been shown to of < 1 mgÆmL)1 [33]. A gel-filtration chromatogram proceed through a green-emitting anionic GFP-like of VFP showed a major peak and a shoulder, indi- intermediate, which has excitation and emission peaks cating a mixture of dimer and monomer species

FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS 1969 Monomeric and dimeric forms of green-emitting FP R. P. Ilagan et al.

(Fig. S1). We therefore sought to design mutations A to shift the equilibrium to a fully monomeric state. With this goal in mind, we aligned the sequences of DsRed and VFP and created a homology model for VFP. B It is known that DsRed forms a strong tetramer, both in solution and in the crystal structure [17,18]. An examination of the crystal structure of the DsRed tetramer shows that the monomers are arranged as a dimer of dimers, with AB (or CD) and AC (or BD) interfaces, as illustrated in Fig. 2A. The AB interface is dominated by hydrophobic interactions, whereas the AC interface is comprised predominantly of salt bridges and hydrogen bonds [19]. Thus, the formation of VFP dimer could be caused by the interaction of either AB or AC. Several point mutations (such as I125R, H162K, A164R and I180T) on the surface of DsRed are documented in the literature, which convert Fig. 2. (A) A cartoon illustration of DsRed tetramer arranged as a the DsRed tight tetramer into a monomer [1,21]. We dimer of dimers with AB (=CD) and AC (=BD) interfaces. (B) compared the residues at these positions in VFP with Structure of two of the four subunits of the tetrameric DsRed consisting of the AC polar interface. The positions of the amino those in DsRed to identify mutations in VFP that acid residues 158 and 160 (corresponding to amino acid residues might shift the monomer–dimer equilibrium towards 162 and 164, respectively, in DsRed) where mutations were monomer. The corresponding amino acid residues in made, are indicated by lines. The chromophore at the center of VFP are H121, N158, T160 and T176, allowing us to the b-barrel structure is shown in black sticks. Protein Data Bank identify possible mutations in VFP as H121R, N158K (PDB) code: 1GGX. [55] and T160R. We focused on examining the N158K and T160R mutations. The locations of these mutations in the AC interface are indicated in Fig. 2B. The ratio- using gel-filtration chromatography. We found that nale for the N158K mutation is that it replaces a polar either the N158K mutation or the T160R mutation is uncharged Asn with a positively charged Lys and this sufficient to convert VFP into an exclusively mono- mutation should disrupt the AC dimerization interface. meric species (Fig. S1). We named these monomeric In DsRed, His162 of the A monomer is involved in N158K and T160R mutants as mVFP1 and mVFP, a stacking interaction with His162 of the adjacent respectively. In the course of the cloning, we also C monomer, whilst simultaneously making an electro- serendipitously isolated the T160A mutant of VFP. static interaction with Glu176 of the C monomer, Gel-filtration chromatography revealed that the T160A forming what appears to be an important part of the mutant is fully dimeric, with no evidence of the mono- AC interface [19]. In VFP, residue 158 (corresponding mer–dimer equilibrium that we observed for VFP to residue 162 in DsRed) is Asn and residue 172 (Fig. S1). Presumably, the introduction of small hydro- (corresponding to residue 176 in DsRed) is Asp. By phobic patches on the surface of the protein promotes contrast, in T160R mutations, the polar uncharged strong dimer formation. We named this dimeric vari- Thr was replaced with the positively charged Arg. In ant of VFP as dVFP. DsRed, position 164 is occupied by Ala, which creates small hydrophobic patches in the AC interface and, by Spectral properties of VFP and its variants replacing it with Arg, the AC interaction is disrupted. Also, previous studies showed that substituting hydro- We proceeded with further characterizations of all four philic or charged amino acids for hydrophobic and proteins, namely VFP and its variants mVFP1 neutral residues of the FP tetrameric interfaces could (N158K), mVFP (T160R) and dVFP (T160A). The generate the monomer form of the protein [13,21]. excitation and emission spectra for all four proteins The mutations were made individually, with the were identical, with an excitation maximum of 491 nm, intention of combining any of them if an individual an emission maximum of 503 nm and a Stokes shift of mutation was insufficient to cause the VFP to mono- 12 nm (Table 1, Fig. 1C and Fig. S2). The measured merize. We expressed and purified each VFP mutant extinction coefficient (EC) of VFP was 83 700 M)1Æ (N158K or T160R) and assessed its oligomeric states cm)1, which was higher than that of EGFP

1970 FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS R. P. Ilagan et al. Monomeric and dimeric forms of green-emitting FP

Table 1. Spectral properties of VFP and its variants in comparison to EGFP, Venus and selected FPs. The excitation (Ex) and emission (Em) wavelengths, the molar extinction coefficients (EC), the quantum yield (QY), the oligomeric states, the relative brightness and the photosta- bility are listed. The relative brightness was calculated from the product of EC and QY. Photostability was calculated based on 100% EGFP measured at the same time. ND, not determined.

Ex Em EC · 10)3 Oligomeric Relative Photostability 1 1 Protein (nm) (nm) (M) Æcm) ) QY states brightness (% EGFP) Reference

VFP 491 503 83.7 1.0 Weak dimer 84 33 This work mVFP 491 503 85.0 0.86 Monomer 73 16 This work mVFP1 491 503 80.4 0.84 Monomer 68 11 This work dVFP 491 503 107.0 1.0 Dimer 107 39 This work EGFP 488 509 54.4 0.60 Monomer 33 100 This work Venus 515 527 93.3 0.75 Monomer 70 27 This work GFPs – Anthozoa AzamiGreen 492 505 55.0 0.74 Monomer 41 ND 44 mWasabi 493 509 70.0 0.80 Monomer 56 53 45 ZsGreen 493 505 43.0 0.91 Tetramer 39 ND 15 copGFP 482 502 70.0 0.60 Tetramer 42 ND 46 cmFP512 503 512 58.8 0.66 Tetramer 39 92 47 aacuGFP2 502 513 93.9 0.71 ND 67 ND 48 aeurGFP 504 515 145.7 0.67 ND 98 ND 48 eechGFP1 497 510 124.2 0.75 ND 93 ND 48 efasGFP 496 507 125.8 0.80 ND 101 ND 48 gfasGFP 492 506 102.5 0.73 ND 75 ND 48 plamGFP 502 514 98.6 0.96 ND 95 ND 48 sarcGFP 483 500 76.7 0.96 ND 74 ND 48 GFP – Aequorea derivatives Superfolder 485 510 83.3 0.65 Monomer 54 90 12 mEmerald 487 509 57.5 0.68 Monomer 39 58 11 Cyan FPs – Anthozoa mTFP1 462 492 64 0.85 Monomer 54 63 49 Photoconvertable FPs Kaede 508 518 98.8 0.88 Tetramer 87 26 50 dEos (G) 506 516 84 0.66 Dimer 55 24 51 mEos2 (G) 506 519 56 0.84 Monomer 47 21 52 Photoswitchable FPs Dronpa 503 517 95 0.85 Monomer 81 ND 53 mKikGR 505 515 49 0.69 Monomer 34 7 54 cerFP505 494 505 54 0.55 Tetramer 30 54 47

(54 400 m)1Æcm)1). The ECs calculated for mVFP1 and The EC and QY for each FP were determined and mVFP were 80 400 and 85 000 m )1Æcm)1, respectively. the product of these two parameters (EC x QY) pro- However, a higher EC value of 107, 000 m)1Æcm)1 was vides the relative brightness (Table 1). We used the observed for dVFP. The increase in the EC value of reported EGFP QY of 0.60 [35] as a reference for cal- dVFP compared with VFP might be caused by its tight culating the QY of VFP and its variants. The relative dimer formation. Table 1 summarizes these data, QY values of VFP and its variants ranged from 0.84 alongside the measured results for EGFP and Venus to 1.0, which was higher than those of both EGFP for comparison. Venus is a variant of yellow FP with and Venus. Thus, as a result of having a high EC and a fast maturation and high brightness [34]. The results a high QY, VFP and its variants produced high rela- obtained for EGFP and Venus are consistent with the tive brightness. It is evident that the dimeric form, values reported in the literature [34,35]. For compari- VFP or dVFP variant, is brighter than the monomeric son, Table 1 includes a list of selected green-emitting form of VFP. Either the mVFP1 or the mVFP variant FPs that have spectral properties relevant to VFP vari- is at least twice as bright as EGFP, and the dVFP var- ants. We reported the fluorescence excitation and emis- iant is much brighter than Venus. To our knowledge, sion wavelength peaks, molar EC, quantum yield there is no monomeric green-emitting FP available to (QY), oligomeric states, relative brightness and photo- date that is at least twofold brighter than EGFP, stability of these selected FPs. except for the photoswitchable Dronpa FP (Table 1).

FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS 1971 Monomeric and dimeric forms of green-emitting FP R. P. Ilagan et al.

We also investigated the pH dependence of VFP A and its variants’ fluorescence emission at 503 nm upon excitation at 491 nm, as shown in Fig. S3. VFP and its variants were found to have pH stability profiles similar to those of EGFP between pH 6 and pH 10.

Fluorescence correlation spectroscopy measurements of size and photostability B We used fluorescence correlation spectroscopy (FCS) to investigate the photobleaching, molecular brightness and oligomeric states of the FPs in more detail. The traces of FCS autocorrelation curves obtained for EGFP and mVFP are shown in Fig. 3A. No shifts in the autocorrelation curves were observed for EGFP as a function of laser power intensity. However, the diffu- sion curves shifted to the left for VFP and its variants Fig. 3. (A) Representative FCS autocorrelation curves of EGFP and as the laser power intensity was increased (Fig. 3A and mVFP taken at increasing laser power intensities from 0.25 to Fig. S4). This shift in autocorrelation curves to the 5 lW. A shift in the autocorrelation curve to the left, to apparently shorter t values, as a function of laser power intensity, was left, noted by shorter apparent diffusion times (tD), is D indicative of photobleaching. observed for VFP and its variants. The autocorrelation curves are normalized to the number of molecules obtained from the fitting The autocorrelation curves for each sample were fit- autocorrelation function [G(t)]. (B) Photobleaching curves for the ted using single-diffusion or two-diffusion component EGFP, Venus, mVFP and dVFP under mercury arc lamp illumination equation. The best-fit curve was assessed based on the using a wide-field microscope. The relative photostability of VFP residual of the fitting. A detailed analysis of the other and its variants are reported in Table 1. photophysical dynamics (e.g. triplet blinking), occur- ring at the submillisecond timescale, is beyond the scope of this paper and will be presented elsewhere. Table 2. Summary of FCS analysis. The autocorrelation curves of each FP obtained at 0.25 lW laser power intensity were fitted The t value and the average fluorescence intensity D using a single-diffusion component equation. The brightness, were determined from the fitting of the autocorrelation expressed as counts per molecule, was calculated by dividing the curves taken at 0.25 lW laser power, as reported in intensity by the number of molecules. Table 2. At this low laser power intensity, the effects of other photophysical processes were minimized. The Counts per Diffusion time Intensity molecule relative molecular brightness of the FPs was calculated FPs (ms) (Hz) 1 · 104 (kHzÆmolecule)1) by dividing the average fluorescence intensity by the number of molecules within the illuminated region. EGFP 0.486 ± 0.012 2.49 ± 0.05 0.262 ± 0.005 The results obtained support our earlier findings that Venus 0.543 ± 0.019 3.25 ± 0.02 0.251 ± 0.002 VFP and its variants are nearly twofold brighter than VFP 0.646 ± 0.004 4.07 ± 0.14 1.76 ± 0.06 mVFP1 0.460 ± 0.007 3.40 ± 0.19 0.485 ± 0.028 EGFP, based on the counts per molecule (kHz ⁄ mole- mVFP 0.472 ± 0.007 3.70 ± 0.14 0.476 ± 0.018 cule) in Table 2 and Table S1. dVFP 0.763 ± 0.004 3.00 ± 0.11 1.55 ± 0.06 At 0.25 lW laser power intensity, the measured rela- tive tD values of either mVFP1 or mVFP were compa- rable to that of the EGFP, indicating that both photobleaching of VFP and its variants in comparison variants are monomeric. Furthermore, VFP and dVFP to that of EGFP and Venus using wide-field microscopy, have tD values greater than that of EGFP, indicating as described in the Materials and methods. Figure 3B higher oligomeric states (Table 2). These results sup- depicts the relative photobleaching curves of EGFP, ported our findings on the oligomeric states of the Venus, mVFP and dVFP from 0 to 500 s. We deter-

VFP variants using gel-filtration chromatography, as mined the relative half time (t½) to photobleach the described earlier. VFP samples, EGFP and Venus (Fig. S5). Based on the

Based on our FCS results, we noticed that photoble- t½ values, we calculated the percentage of photostability aching occurs in VFP and its variants. This observation of the VFP and its variants relative to 100% EGFP. We prompted us to investigate, in greater detail, the rate of also included, in Table 1, the reported photostability of

1972 FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS R. P. Ilagan et al. Monomeric and dimeric forms of green-emitting FP some FPs relative to 100% EGFP measured at the same A time. The photostability data of other green-emitting FPs have not yet been reported or determined. The mVFP1 and mVFP variants, which have 11% and 16% photostability, respectively, were less photostable than VFP and dVFP. However, the dVFP variant has 39% photostability, and thus exhibits greater photostability than Venus and other photoconvertable or photoswitch- able FPs. For other imaging applications [24], the differ- ence in photostability has no relevance. Even with this photostability, our VFP variants can be useful for numerous in vitro and in vivo detection applications.

B Application of VFP variants as detection markers It has been shown previously in our laboratory that a protein recognition domain, tetratricopeptide repeats (TPR), fused to EGFP can be used to detect the pro- tein–peptide interaction in a single step, completely eliminating the use of primary and secondary antibodies in western blot analysis [36]. The TPR-based recognition module (T-Mod) was demonstrated to bind specifically to MEEVF peptide fused to glutathione S-transferase (GST) [36]. The fusion of FP to T-Mod can completely eliminate the need for any antibodies or developing pro- cedures, which makes western blotting faster, simpler and less costly. We adapted this experiment to show the Fig. 4. (A) Comparison of the T-Mod fused to EGFP, mVFP, or dVFP usefulness of mVFP and dVFP brightness in compari- as a replacement for antibodies in western blot analysis. A duplicate son to EGFP. We expressed and purified the T-Mod SDS-polyacrylamide gel used in western blotting was stained with fused to EGFP, mVFP or dVFP. Following the Coomassie Brilliant Blue. Lanes 1, precision plus protein standard )1 SDS ⁄ PAGE of E. coli-expressing GST–MEEVF lysate, (BioRad); lane 2, lysate; lane 3, lysate supplemented with 1 mgÆmL of purified GST–MEEVF; lane 4, purified GST–MEEVF. The arrow gels were transferred to poly(vinylidene difluoride) indicates the GST–MEEVF protein band. (B) Microinjection of membrane and processed as for western blotting. After KH–mVFP fusion mRNA into zebrafish embryos. The expression blocking the membrane, we incubated the blots sepa- of KH–mVFP protein was monitored in the embryos at 6 and 14 hpf rately with different T-Mod–FPs for 1 h at room tem- using fluorescence microscopy. Zebrafish embryos without RNA perature. The membrane was then visualized using a UV injections were used as a control. transilluminator at 302 nm, as shown in Fig. 4A. The visible band indicated by an arrow is the GST–MEEVF protein detected by the binding of T-Mod–FP. The the KH–mVFP fusion protein into zebrafish embryos bands from T-Mod–mVFP or T-Mod–dVFP were at at the one-cell stage. Live embryos at 6-h post-fertiliza- least two-fold brighter than that of the EGFP. Addi- tion (hpf) and at 14 hpf (10-somite) stages were tional bands were visible in the membrane incubated mounted on glass slides and visualized using a fluores- with T-Mod–dVFP as a result of the intense brightness cence microscope, as shown in Fig. 4B. The fluores- of the dVFP protein. This result illustrates the benefit of cence signals from zebrafish embryos with KH–mVFP having high brightness, in terms of sensitivity, in a prac- were more intense than those of the control, which tical detection application. showed a faint cellular autofluorescence.

Application of mVFP as an in vivo marker Discussion To demonstrate that our VFP can be used for in vivo We have described a detailed characterization of a new labeling, we chose the monomeric form, mVFP, and FP from the warm water coral, C. microphthalma, fused it to the KH domains of fragile X mental retar- collected off Lizard Island on the Australian Great dation protein (FMRP). We injected mRNA encoding Barrier Reef. The protein, which we named VFP,

FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS 1973 Monomeric and dimeric forms of green-emitting FP R. P. Ilagan et al. matures rapidly at 37 °C and emits bright green fluo- Sequence alignment and homology modeling rescence. VFP, as isolated, showed a propensity to form fairly weakly associating dimers. By creating a Sequence alignment of VFP with EGFP and DsRed was homology model of VFP, we were able to create sur- performed using clustalw2 (EMBL-EBI). Homology face mutations that convert VFP into either an exclu- modeling was carried out using swiss-model [38]. sively monomeric species (N158K or T160R) – which we named mVFP1 and mVFP, respectively, or into an Recombinant protein expression exclusively dimeric species (T160A) – which we named dVFP. This rational approach to creating monomeric The proteins were expressed in E. coli DH10b cells grown in variants can be used as a guide for re-engineering Luria–Bertani (LB) liquid medium for 24 h at 37 °C. The cells other coral FPs that have higher oligomeric forms. were harvested by centrifugation and the pellets were resus- These novel proteins have features that will be useful pended in lysis buffer (50 mm Tris ⁄ HCl, pH 7.4, 300 mm for a variety of applications. The mVFP1 and mVFP NaCl) supplemented with a tablet of complete EDTA-free variants are both monomeric and fluoresce at least twice protease inhibitor cocktail (Roche) and 5 mm b-mercapto- as brightly as EGFP. The dimeric dVFP is even brighter, ethanol. The lysate was sonicated, then centrifuged. The supernatant solution was loaded into Ni-nitrilotriacetic acid being at least 1.5 times as bright as Venus. For applica- agarose (Qiagen, Valencia, CA, USA), and the pure protein tions where oligomerization is not critical, the use of the was eluted with 50 mm Tris ⁄ HCl, pH 7.4, 150 mm NaCl, dVFP variant would be advantageous because of its 200 mm imidazole. The fractions containing the protein were high brightness. When a bright, monomeric protein is pooled and dialyzed into 50 mm Tris ⁄ HCl, pH 7.4, 150 mm desired, mVFP1 or mVFP would be the proteins of NaCl. The purity of the samples was determined by choice. Based on the list of reported FPs (either wild- SDS ⁄ PAGE. The proteins were concentrated by centriprep type or engineered) (Table 1), none is both monomeric YM-10 with 10 000 MWCO (Amicon, Billerica, MA, USA) and at least two-fold brighter than EGFP, except for to about 100–200 mm then stored in aliquots at )20 °C. The photoswitchable Dronpa. The data we presented should buffer used in all spectroscopic analyses was 50 mm allow investigators to choose which VFP variant is the Tris ⁄ HCl, pH 7.4, 150 mm NaCl, unless otherwise noted. most appropriate for their specific research application. With regards to photostability, VFP and its variants photobleached at a faster rate than EGFP. The vast Analytical gel-filtration chromatography majority of reports in the literature describing green- The molecular sizes of the purified FPs were analyzed using a emitting FPs isolated from corals do not include Superdex S200 10 ⁄ 30 gel-filtration column (Amersham Phar- photostability measurements, which makes it difficult macia) by FPLC at room . A 100 mL sample of to assess the level of photostability of VFP variants in < 0.01 mgÆmL)1 of each FP was injected into the column at relation to other coral FPs [2,24]. However, for many a flow rate of 0.5 mLÆmin)1 and the absorbance was moni- imaging applications, this photobleaching property will tored at 280 nm. The oligomeric states of the VFP and its not be influential [24,34]. In conclusion, the monomeric variants were determined based on the EGFP elution time and the dimeric forms of VFP represent viable alterna- and protein standards (Bio-Rad, Hercules, CA, USA). tives to the widely used EGFP and Venus. Absorption spectroscopy Materials and methods The absorbance spectra of the FPs were recorded on a Hewlett Packard 845X UV-visible Chemstation. The ECs Plasmid constructions and mutations of the FPs were calculated based on the absorbance of the The plasmids encoding VFP, EGFP and Venus with poly- native and acid-denatured or alkali-denatured proteins. The histidine tags were constructed as previously described ECs of the GFP-like chromophores used in the calculation )1 )1 [26,37]. The VFP coding sequence was deposited in the are 44 000 m Æcm at 447 nm in 1 m NaOH [33] and )1 )1 EMBL nucleotide sequence database under the accession 28 500 m Æcm at 382 nm in 1 m HCl [39]. For yellow FP, number FN597286 and in the UniProtKBT TrEMBL pro- Venus, the EC of the chromophore was back-calculated ⁄ )1 )1 tein sequence database under the accession number D1J6P8. using 22 000 m Æcm at 280 nm in 10 mm Tris ⁄ HCl. Site-directed mutagenesis (QuikChange Site-Directed Muta- genesis Kit; Stratagene, Cedar Creek, TX, USA) was used spectroscopy to introduce the N158K and T160R mutations into VFP. Mutations were verified by DNA sequencing (W. M. Keck, Fluorescence excitation and emission measurements were Foundation Facility, Yale University, CT, USA). performed using a PTI Quantamaster C-61 two-channel

1974 FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS R. P. Ilagan et al. Monomeric and dimeric forms of green-emitting FP

fluorescence spectrophotometer. The samples were excited (Pierce, Rockford, IL, USA) and shaking, with occasional at 450 nm and emission spectra were measured from 465 to vortexing, for 10 min. The lysate was supplemented either 650 nm with a 2 nm slit-width. Fluorescence excitation with or without 1 mgÆmL)1 of purified GST–MEEVF pro- spectra were obtained from 250 to 515 nm by monitoring tein. The samples mixed with a reducing loading buffer the emission at 530 nm with a 2 nm slit-width. The QY were loaded precisely into 4–12% gradient SDS-polyacryl- values of the VFP and its variants were determined relative amide gels together with an equivalent amount of purified to EGFP (QY = 0.60 [35]). The pH dependence of VFP GST–MEEVF protein. The gels were run at room temper- and its variants’ fluorescence emission at 503 nm were ature for 1 h at a voltage of 120 V using NuPAGE buffer measured upon excitation at 491 nm at room temperature. (Invitrogen, Carlsbad, CA, USA). One gel was stained pH titrations were performed using a series of 100–200 mm with Coomassie Brilliant Blue while the other gels were citrate-phosphate buffer (pH 2.0–11.0) containing 150 mm transferred onto a poly(vinylidene difluoride) membrane NaCl. (Millipore, Billerica, MA, USA). Membrane transfer was carried out in a cold room for 3 h at a constant current of 380 mAmp. The transfer buffer used contained 24 mm FCS Tris-base, 192 mm glycine, 10% methanol and 0.01% FCS measurements were made on a laboratory built instru- SDS. The membranes were blocked in 5% non-fat milk in ment, based around an inverted microscope with a 488 nm TBS-T (20 mm Tris-base, pH 8.0, 150 mm NaCl, 0.1% DPSS laser for excitation, as previously described [40,41]. Tween-20) overnight at 4 °C with shaking. The mem- All measurements were carried out on FP samples of branes were then incubated individually with each 5 lm approximately 100 nm using varying laser power intensities T-Mod-FP fusion construct in TBS-T containing 0.1% from 5 to 0.25 lW measured on the table before entering nonfat milk for 1 h at room temperature with shaking. the microscope. The output of the detection channels was The membranes were washed three times with TBS-T, for autocorrelated in a digital correlator (Correlator.com). 10 min each wash, visualized using a UV transilluminator Control measurements were performed using Alexa 488 at 302 nm and the images captured using a to ensure the proper alignment of the confocal (Kodak, Rochester, NY, USA). optics and the absence of artifacts in the FCS. The autocor- relation curves were fitted using a single- or two-component equation, as previously described [41]. The parameters mRNA microinjection assay extracted from the fittings were relative tD number of mole- To assemble the KH–mVFP fusion construct, the deleted cules, and fluorescence intensities. KH domain of human FMRP – hFMRP(KH1-KH2D)– was fused with the N-terminus of mVFP and cloned into Photobleaching the mammalian PCS2 + vector. The construct was sequenced (W. M. Keck Foundation Facility, Yale Univer- Photobleaching measurements of purified FP samples were sity) and named KH–mVFP for simplicity. The in vitro performed using a inverted wide-field microscope equipped synthesis of large amounts of capped RNA was carried with a 100 W mercury arc lamp similar to those described out using the mMESSAGE mMACHINE kit (Applied in the literature [42]. The FP samples were mixed with min- Biosystems ⁄ Ambion, Austin, TX, USA) following the eral oil, and about 5 lL of the mixture was sandwiched manufacturer’s protocols. The capped transcription reaction between a glass slide and a cover slip. A neutral density was prepared at room temperature and then incubated at filter was used initially for sample alignment, which was 37 °C for 2 h. TURBODNase (Ambion) was added to the removed when the actual measurements were being reaction and incubated at 37 °C for another 15 min to made. The FP samples were imaged using a 50 ms exposure remove the template DNA. The RNA was purified using time and a frame rate of one image per second. The the RNeasy Mini kit (Qiagen). The of the measurement was taken in a 600 s time span under RNA was determined using a UV-vis spectrometer and constant illumination. then the RNA was stored at )80 °C until use. The RNA microinjections were performed at the one-cell Western blot assays stage using standard protocols [43]. The injection solution consisted of 200 ngÆlL)1 of KH–mVFP and 0.15% The T-Mod–FP and GST–MEEVF constructs were pre- Phenol Red in Danieau’s solution. Live embryos at 6 pared as previously described [36]. The FP fused to and 14 hpf stages were manually dechorionated and T-Mod was EGFP, mVFP, or dVFP. Each construct was mounted in methylcellulose. In parallel, we also mounted transformed into E. coli BL21(DE3) cells and the protein embryos without RNA injections as a control. Fluorescent was purified following the protocol previously described images were acquired on a Zeiss Axioskop microscope [36]. The GST–MEEVF lysate was obtained from 6-mL using a 20 · objective and an FITC filter. Color adjustment overnight culture cell pellet by adding 1 mL of B-Per of the fluorescent images was made equally for both

FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS 1975 Monomeric and dimeric forms of green-emitting FP R. P. Ilagan et al.

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52 McKinney SA, Murphy CS, Hazelwood KL, Davidson Fig. S2. Absorption, fluorescence excitation and emis- MW & Looger LL (2009) A bright and photostable sion spectra of VFP and its variants. photoconvertible fluorescent protein. Nat Methods 6, Fig. S3. pH dependence of the fluorescence of VFP 131–133. and its variants in comparison with EGFP and 53 Ando R, Mizuno H & Miyawaki A (2004) Regulated Venus. fast nucleocytoplasmic shuttling observed by reversible Fig. S4. Representative FCS autocorrelation curves of protein highlighting. Science 306, 1370–1373. Venus, VFP, mVFP1 and dVFP taken at increasing 54 Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A & laser power intensities from 0.25 to 5 mW. van Oijen AM (2008) mKikGR, a monomeric photo- Fig. S5. Photobleaching curves for EGFP, Venus, switchable fluorescent protein. PLoS ONE 3, e3944. VFP and its variants under mercury arc lamp illumi- 55 Wall MA, Socolich M & Ranganathan R (2000) The nation. structural basis for red fluorescence in the tetrameric Table S1. Summary of fluorescence correlation spec- GFP homolog DsRed. Nat Struct Biol 7, 1133–1138. troscopy (FCS) analysis. This supplementary material can be found in the Supporting information online version of this article. Please note: As a service to our authors and readers, The following supplementary material is available: this journal provides supporting information supplied This section includes the complete set of character- by the authors. Such materials are peer-reviewed and ization of VFP and its variants in comparison to may be re-organized for online delivery, but are not EGFP and Venus: copy-edited or typeset. Technical support issues arising Fig. S1. Gel filtration chromatography of VFP, mVFP, from supporting information (other than missing files) dVFP, and EGFP using Superdex S200 10 ⁄ 30 gel fil- should be addressed to the authors. tration column in FPLC at room temperature.

1978 FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS