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1 VALWAY Technology Center, NEC Soft, Ltd, 1-18-7, Shinkiba, Koto-ku, Tokyo 136-8627, Japan
2 National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
3 Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0202, USA
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Abstract |
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ex = 509 nm, em = 517 nm) compared with those of GFP from Aequorea victoria (avGFP) and other GFP-like proteins from marine copepods. We performed crystallographic and biochemical studies to understand why this shift occurs in CpYGFP. The structure of CpYGFP showed that the imidazole side chain of His52 is involved in stacking on the phenol moiety of the chromophore. We investigated the potential role of His52 in causing the red-shifted spectral properties by performing mutational analyses of H52T, H52D and H52F. The emission wavelengths of H52T and H52D were blue-shifted and that of H52F was red-shifted relative to the wild type. Comparison of its structure of another copepod GFP (ppluGFP2) having an emission maximum at 502 nm showed that the imidazole ring of His54 (corresponding to His52 in CpYGFP) is flipped out of the stacking position with the chromophore. These findings suggest that 
– stacking interaction between His52 and the phenol moiety of the chromophore is the likely cause of the red-shift in light emission. |
Introduction |
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Six GFP homologs comprise a new class of avGFP from marine copepods of the family Pontellidae (Shagin et al. 2004) and a homologous protein emitting novel yellowish-green fluorescence has been cloned from the marine copepod Chiridius poppei (CpYGFP) (Masuda et al. 2006). The expressed protein has excitation and emission maxima at 509 and 517 nm, respectively, which are significantly longer than those of avGFP (395 (475) and 508 nm, respectively) (Morise et al. 1974), but much shorter than those of drFP583 (558 and 583 nm) from Discosoma sp. (Matz et al. 1999), which is commercially available as DsRed. Furthermore, CpYGFP has significantly red-shifted spectra compared with other copepod GFPs despite having high amino acid sequence similarity. The crystal structure of one copepod GFP from the planktonic copepod Pontellidae plumata (ppluGFP2) has been determined (Wilmann et al. 2006) and its engineered protein commercially available as TurboGFP has also been determined (Evdokimov et al. 2006). Here, we used X-ray crystallography to analyze the structure of CpYGFP and then compared it with those of ppluGFP2 and TurboGFP to determine the structural basis for the red-shift in CpYGFP. These investigations revealed a novel stacking interaction between His52 and the phenol moiety of the chromophore. We found that His52 is the fourth residue preceding Tyr56 of the tri-peptide sequence of the chromophore. We also performed mutational studies to confirm whether this stacking interaction is important to determining the optical properties.
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Results |
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While the primary structure of CpYGFP had only 21% identity with DsRed (Fig. 1), the latter functioned well as an initial model during MR calculations and two monomers were identified in an asymmetric unit. Residues 2–218 of one monomer (Monomer A) and residues 2–217 of another (monomer B) were assigned to an electron density map. The first methionine, which could not be found in the map, appeared to be digested because the next residue was threonine. The final model contained one each of monomers A and B, one CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) and 437 water molecules. The CAPS molecule in the crystal played an important role as an additive for packing insofar as the space group of C2221 would change to P213 with a much larger unit cell in its absence (data not shown). The CAPS molecule was located between monomers A and B (monomer B and symmetry-related monomer A in PDB code 2DD7) and it interacted with both monomers (Supporting Fig. S1). The cyclohexyl ring of CAPS interacted with a hydrophobic patch formed by Pro124 and Tyr164 and the methylene chain of the Glu90 of monomer B, and the positive charge of the nitrogen atom of CAPS interacted with the Glu90 of monomer B. This nitrogen atom also interacted with the Asp160 of monomer A, and the sulfonic acid moiety in CAPS interacted with Thr129 and Lys127 of monomer A. This CAPS-induced lattice appeared to result in a rather low averaged B-factor (Table 2) despite the high solvent content (70%).
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superpositions between the two monomers in an asymmetric unit was 0.42 Å for all residues from 2 to 217. Loops flipped in residues from 143 to 146 of one monomer, but not in the other. If the residues from 143 to 146 were not included, then the rms difference decreased to 0.21Å. For simplicity, the following discussion is based on monomer A, unless noted otherwise. The secondary structure of monomer A is β1 (4–14), β2 (17–28), β3 (31–39), 1 including a chromophore region (47–61), βextra (63–64), 2 (71–77), β4 (80–89), β5 (93–104), β6 (107–117), β7 (132–143), β8 (147–158), β9 (162–174), β10 (188–199), and β11 (202–212) (Fig. 1) and folded into a β-barrel motif comprising 11 β-strands and a central helix (Fig. 2a,b), which is a characteristically common fold among GFP-like proteins. The overall folding of CpYGFP is similar to that of other GFP-like proteins, where 11 β-strands enclose a central helix with a chromophore to form a β-barrel structure that is highly conserved (Fig. 2b). For example, the rms difference in the C superposition including all strands and helices between CpYGFP and DsRed (Wall et al. 2000), avGFP (Örmo et al. 1996) or ppluGFP2 (Wilmann et al. 2006) is 0.93, 1.24 or 0.94 Å, respectively. To correctly align the amino acid sequences except for ppluGFP2 is difficult before structural determination even using sequence alignment programs because the amino acid sequence identities of CpYGFP to DsRed and avGFP are only 21.0% and 15.5%, respectively, despite the structural similarities. Structure-based sequence alignment (Fig. 1) shows that insertions, deletions and low amino acid identities are apparent in the region from 176 to the C-terminus (CpYGFP numbering) compared with other regions. In particular, a coil-like loop between β9 and β10, which lies near 2 and covers the top of the β-barrel (Fig. 2b), has the most divergent feature among these GFP-like proteins. The conformation at the C-terminal end of this loop in CpYGFP appears to contribute to enlargement of the contact surface for dimerization. On the other side of the β-barrel, the conformation of a loop around residue 200 (CpYGFP numbering) is variable, but this loop of CpYGFP is cut short, and it forms the most compact fold of this region among GFP-like proteins (Fig. 2b).
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All known GFP-like proteins form monomers, dimers or tetramers. For example, avGFP is monomeric in aqueous solution, but dimeric at high concentrations and under specific solvent conditions. Crystal structures of avGFP have also been solved as monomers (Örmo et al. 1996) and dimers (Yang et al. 1996). On the other hand, the red fluorescent DsRed and eqFP611 proteins and nonfluorescent Rtms5 proteins form tetramers, which have a similar 222-symmetry fold through two independent interacting surfaces (Wall et al. 2000; Petersen et al. 2003; Prescott et al. 2003). The present crystal structure of CpYGFP is dimeric, which is in agreement with the value of the molecular mass estimated by gel filtration column chromatography (Masuda et al. 2006). Hydrophobic interaction is mainly involved in CpYGFP dimerization. A region consisting of Val137, Phe189, His191 and Phe212 (highlighted with yellow in Fig. 2c) forms the main part of the hydrophobic interface, which interacted with the same region on the neighboring monomer. Another region consisting of Cys134, Pro135, Ala155 and Phe163 interacts with a region including Leu139, Leu141 and Pro187 of the neighboring monomer (highlighted with green in Fig. 2c). Such relative positions of monomers in a dimer of CpYGFP appear different from those of the avGFP dimer (Yang et al. 1996), but similar to those of the ppluGFP2 dimer (Wilmann et al. 2006) and also to those in a dimer within the DsRed tetramer (Wall et al. 2000) as follows. The DsRed tetramer has two independent dimer-interfaces within the 222-symmetry. One is between monomers A and B, and the other is between monomers A and C (Fig. 2d). The former is involved in hydrophilic interactions including water-mediated hydrogen bonds and the latter is involved in hydrophobic interactions mainly due to valine and isoleucine residues. When monomer A of CpYGFP was fitted to monomer A of DsRed, monomer B of CpYGFP was superposed very closely to monomer B but not to monomer C of DsRed (Fig. 2d). This means that the hydrophobic dimer-interface in CpYGFP is replaced by the hydrophilic dimer-interface in DsRed. This is understandable because most of hydrophobic residues participating in dimerization in CpYGFP are replaced by hydrophilic residues in DsRed as shown in Fig. 1. Therefore, the relative position of the dimer can be considered as highly conserved, whereas the interaction manner of dimerization is not, as far as CpYGFP and DsRed are concerned.
Chromophore structure and environment
The chromophore of CpYGFP consists of a Gly55-Tyr56-Gly57 tri-peptide sequence. Around the chromophore, only Arg85 and Glu207 are conserved, as found in other GFP-like proteins, and they are considered to be involved in the catalysis of chromophore formation, specifically in cyclization and/or oxidation (Matz et al. 1999). The side-chain amino group of Arg85 hydrogen bonds to the imidazolinone carbonyl group of the chromophore, while the carboxyl oxygen atom of Glu207 hydrogen bonds to the imidazolinone amino group of the chromophore. The hydroxyl group of the phenol moiety of the chromophore forms a hydrogen bond with the side-chain oxygen atom of Thr136. This hydrogen bond is conserved in ppluGFP2 and TurboGFP. In avGFP, Thr136 is not conserved, but Thr203 might be displaced to maintain the hydrogen bond to the hydroxyl group of the phenol moiety. Water molecules also contribute to stabilization of the chromophore structure, and two of them are involved in direct hydrogen bonding with the chromophore (Fig. 3a). The most notable structural feature around the chromophore is that His52 is stacked on its phenol moiety (Supporting Fig. S2). The shortest atom–atom distance between stacked planes is 3.14 Å in monomer A and 3.18 Å in monomer B. His52 is also stacked on the guanidino group of Arg154, forming a stable sandwiched structure. His52 in CpYGFP is positioned at the fourth residue preceding Tyr56 in the chromophore. If the residues from His52 to Tyr56 were to participate in a typical -helix, His52 could not stack on the phenol moiety because the pitch of a typical -helix is known to be 3.6. Hence, the central helix of GFPs have an unusual pitch at the chromophore as a result of an autocatalytic cyclization on the main chain, which then might allow His52 stacking to occur on the chromophore. The residue corresponding to His52 is threonine in some GFP-like proteins including avGFP, or proline in some other GFP-like proteins including DsRed. These residues could not participate in stacking interaction with the phenol moiety of the chromophore. A stacking interaction also occurs in the quadruple mutant T203Y/S65G/V68L/S72A of GFP, referred to as YFP (Wachter et al. 1998), where the phenol side-chain of Tyr203 is the stacking residue, although Tyr203 is located at the opposite side relative to the phenol plane of the chromophore in contrast with the case of CpYGFP.
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Whether or not the wavelength shift is caused by a stacking interaction of His52 was examined by mutation experiments. The reasons why we selected mutant H52T, H52F and H52D proteins were as follows. Threonine in H52T can serve as a representative residue of alanine, valine, leucine, isoleucine, methionine and threonine, which should have no – stacking on the phenol moiety of the chromophore. H52F could confirm stacking effects on the phenol moiety. The tyrosine residue was assumed to behave like phenylalanine. We used aspartic acid in H52D to examine the effect of a negative electrostatic charge on the phenol moiety of the chromophore. Glutamic acid has a longer side chain, which could interfere with the negative charge being able to ride on the phenol ring of the chromophore. Table 1 and Fig. 4c shows the fluorescence spectra of the mutants. The emission maximum of H52T being 511 nm indicated a significant blue-shift, whereas that of H52F was at 522 nm, indicating more of a red-shift than that of the wild type. The blue-shift emission might have been due to the absence of a stacking interaction with the chromophore, and red-shift might be attributable to the amount of overlapping -electrons of phenylalanine being greater than that of histidine. The spectrum contained a shoulder peak for the wild type at 475–485 nm and for H52F at 480–490 nm, but not for H52T and H52D. This indicates a relationship between the shoulder and the stacking interaction. A shoulder at 480–490 nm is also found in the spectrum of YFP, which has a stacking interaction (Wachter et al. 1998).
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positions was < 0.2 Å. A single difference is located around the mutated residue near the chromophore (Fig. 3b). Thr52 appears to form CH- interaction with the phenol moiety of the chromophore in place of – interaction. Superposition with the wild type (Fig. 3b) revealed that Arg154 moves close to the chromophore, as if to fill a void created by the replacement of threonine with its smaller side chain. The phenol moiety of the chromophore also moved close to residue Thr52 through a slight tilt (4.5°) of the phenol ring. No other changes were evident. Thus, the structural and mutational studies provided support for the hypothesis that His52 stacking on the phenol moiety of the chromophore induces a red-shift in emission wavelength. |
Discussion |
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In comparison with previous findings of yellow fluorescent proteins, the phenol moiety of the chromophore of CpYGFP has a novel stacking interaction with His52, which is located four residues before Tyr56 in the chromophore. His52 is not conserved except in two copepod GFPs (ppluGFP2 and TurboGFP), although these proteins emit green fluorescence. This difference in optical properties among copepod GFPs can be explained by the structural comparison shown in Fig. 3c,d. In ppluGFP2, His54 (corresponding to His52 in CpYGFP) is flipped by rotating the C-Cβ bond compared with His52 in CpYGFP. This flip disrupts the stacking interaction with the phenol moiety of the chromophore that is essential for red-shifted emission. The bulky side chain of Phe168 in ppluGFP2 (corresponding to residue Ala166 in CpYGFP) is inserted between Glu89 and Arg156, disrupting the salt bridge between the two and causing them to move apart. The flipping of His54 appears to be induced to avoid contact with the rearranged Arg156. The flipped His54 participates in a new hydrogen bond with Glu89 (Fig. 3c). Phe168 with a bulky side chain is replaced with Ser in TurboGFP (Evdokimov et al. 2006). However, the structural rearrangement including His54 flipping is similar even with this replacement (Fig. 3d). Such structural rearrangement caused by the amino acid displacement might be due to the tight packing around Ala166 in CpYGFP.
Figure 4c and Table 1 show that the wild type has a single lower energy peak as well as H52T or H52F, whereas H52D has two excitation maxima at 408 and 499 nm. Two similar absorption maxima have been identified for avGFP at 395 and 475 nm (Morise et al. 1974); the former and latter wavelengths may be ascribed to the neutral and anionic states of the chromophore respectively but the mutation of avGFP (S65T) completely suppresses the 395 nm peak. A structural comparison of avGFP and the S65T mutant suggested that the equilibrium between the anionic and neutral states is governed by a hydrogen bonding network that permits proton transfer between the chromophore and neighboring side chains (Brejc et al. 1997; Shu et al. 2007). The present findings indicated that the chromophores of the wild type, H52T and H52F should be anionic. Hydrogen bonding networks (Fig. 5) suggest that the hydroxyl group of the phenol moiety is deprotonated and that it acts as a proton acceptor from Thr136. Each of Arg154 and Arg192 participates in hydrogen bonding networks and also functions as a positively charged residue that is important for electrostatic interaction with the anionic phenol moiety. The positively charged environment formed mainly by Arg154 and Arg192 contributes to stabilization of the anionic form of the chromophore. The above networks and arginine residues are conserved in ppluGFP2 and TurboGFP. This perspective is consistent with that the estimated pKa is 
4.6 based on apparent titration curves generated from absorbance scans (Fig. 4b) and the reported pKa for ppluGFP2 is 4.3, which is the lowest value for a GFP that can be excited at ~488 nm (Wilmann et al. 2006). Figure 4b shows that the wild type of CpYGFP has one absorbance peak at 508 nm when the pH is > ~4.8 in the titration curve, which reflects that the chromophore is in a highly stabilized anionic state. This is in contrast to the spectral properties of YFP that are highly sensitive to pH change (Wachter et al. 1998). On the other hand, H52D has two excitation peaks (Table 1), suggesting the presence of two different forms caused by the neutral and anionic states of the chromophore, as in the case of avGFP mentioned above. The protonated form of the phenol moiety of the neutral chromophore might be occurred even in the wild type under acidic condition because of observation of small peaks around 400 nm under pH4.6 and pH4.8 (Fig. 4a).
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Experimental procedures |
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Recombinant CpYGFP was expressed and purified as described (Masuda et al. 2006). Plasmids overexpressing CpYGFP mutants (H52T, H52F and H52D) were prepared by site-directed mutagenesis and expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL Competent Cells (Stratagene). Protein samples were purified by anion exchange column chromatography, using HiTrap DEAE (GE Healthcare), and the absorbance of the eluants was monitored at 490 nm. The proteins were concentrated using VIVAspin (Sartorius) and diluted with 20 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl. Emission spectra were measured using an F-4500 fluorescence spectrometer (Hitachi) at 480 or 470 nm, whereas excitation spectra were measured at 520 (wild type and H52D), 518 (H52T) and 530 (H52F) nm. The fluorescence spectra in Fig. 4c are normalized from their peaks, and Table 1 summarizes the peak wavelengths for each protein. Protein solutions used for crystallization were further purified using Superdex 200 10/30 GL gel filtration and by anion exchange chromatography, using MonoQ (GE Healthcare).
Crystallization and structure determination
Purified CpYGFP was crystallized by sitting-drop vapor diffusion at 18 °C. Drops comprised 1 µL of 20 mM Tris-HCl (pH 8.5) containing 80 mM NaCl and 23 mg/mL of CpYGFP with 1 µL of reservoir solution. Rod-shaped crystals formed in reservoir solution comprising 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) (pH 10.5), 0.2 M Li2SO4 and 2.2 M (NH4)2SO4. Diffraction data were collected under cryogenic conditions at 100 K under a stream of nitrogen at beamline BL-6A of the Photon Factory (PF; Tsukuba, Japan) using an ADSC Quantum-210 CCD detector. The cryoprotectant comprised a mixture of glycerol and reservoir solution. The space group was C2221 with a unit cell of a = 113.5, b = 133.5 and c = 108.7 Å, which corresponded to VM = 4.2 Å3/Da (solvent content 70%) when the asymmetric unit contained two monomers. The reflection data to 1.9Å resolution was processed using the MOSFLM program (Leslie 2006). We used 20 mM Tris-HCl (pH 8.5), containing 100 mM NaCl and 8 mg protein/mL to crystallize H52T, and the optimal reservoir solution, 0.1 M CAPS (pH 10.5), containing 0.2 M NaCl and 2.6 M (NH4)2SO4. Data sets were collected using an ADSC quantum 210 CCD detector at beamline NW12, PF-AR (Tsukuba, Japan). The space group was P212121 with the unit cell being a = 108.7, b = 114.0 and c = 133.4 Å. The unit cell constants were similar to those of wild-type crystals except the crystal had another space group. The VM value was 4.2 Å3/Da when the asymmetric unit contained four monomers. Table 2 summarizes the data collection statistics.
Initial phases of CpYGFP were calculated using molecular replacement (MR). A search model was constructed based on coordinates of DsRed deposited in the Protein Data Bank with an ID of 1GGX [PDB] (Wall et al. 2000). We performed structural refinements using the CNX (Brunger et al. 1998) and XtalView programs (McRee 1999). We omitted 5% of the reflection data when calculating the free R-factor during refinement. The final Rwork was 20.4% (Rfree = 23.1%). A phase set for the mutant crystal was calculated using MR and the structure of wild-type CpYGFP to refine the model. Table 2 summarizes the refinement statistics. The atomic coordinates and structure factors for wild-type CpYGFP (code 2DD7) and the H52T mutant (code 2DD9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org).
Measurement of absorbance spectra
Concentrated CpYGFP in 5 mM HEPES-NaOH (pH 7.3) and 0.1 M NaCl was diluted into measurement buffers comprising 0.1 M acetate-NaOH (pH 4.6, pH 4.8, pH 5.0, pH 5.2), MES-NaOH (pH 6.0), Tris-HCl (pH 7.0, pH 8.0) or Bicine-NaOH (pH 9.0) and 0.1 M sodium chloride. CpYGFP and measurement buffer mixed at a ratio of 14:3 were incubated at room temperature for 40 min and then absorbance spectra were measured using an Ultrospec 2100 pro (GE Healthcare).
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Acknowledgements |
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Footnotes |
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* Correspondence: mizuno-hiroshi{at}aist.go.jp
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References |
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Accepted: 13 March 2009
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