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Logo of actad2this articlesearchopen accesssubscribesubmitActa Crystallographica Section D: Biological CrystallographyActa Crystallographica Section D: Biological Crystallography
 
Acta Crystallogr D Struct Biol. 2016 August 1; 72(Pt 8): 922–932.
Published online 2016 July 13. doi:  10.1107/S205979831601038X
PMCID: PMC4973210

Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms

Abstract

The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβ bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

Keywords: fluorescent photoconvertible proteins, green fluorescent protein, red fluorescent protein, three-dimensional structure, chromophore, β-barrel, structure–function relationships, DendFP, DendRFP, DendGFP, Dendronephthya

1. Introduction  

Fluorescent proteins (FPs) from the GFP family have become indispensable noninvasive tools for imaging live cells, tissues and entire organisms. Among them, phototransformable fluorescent proteins (PTFPs) form a distinct class of FPs, the spectroscopic properties of which can be controlled by light (Chudakov et al., 2010  ; Piatkevich & Verkhusha, 2010  ; Wiedenmann et al., 2009  ; Nienhaus et al., 2005  ). PTFPs comprise of three main groups: photoactivatable fluorescent proteins (PAFPs), photoconvertible fluorescent proteins (PCFPs) and reversibly switchable fluorescent proteins (RSFPs). PAFPs are proteins that undergo an irreversible light-induced activation from a nonfluorescent state to a fluorescent state (Patterson & Lippincott-Schwartz, 2002  ). PCFPs, on the other hand, exhibit an irreversible conversion between two fluorescent states, whereas RSFPs can be photoswitched back and forth between a fluorescent ON state and a nonfluorescent OFF state (Chudakov et al., 2003  ; Ando et al., 2004  ). The phototransformations of PAFPs and PCFPs involve covalent modifications of the FPs, while RSFPs undergo only conformational rearrangements of the chromophore and its immediate environment.

Because of the ability of PTFPs to change their fluorescence upon exposure to light of a specific wavelength, they have become essential marker tools for live-cell optical imaging with super-resolution. PAFPs are ideal for regional optical marking in pulse-chase experiments on live cells and tissues. PSFPs can be used in patterned illumination microscopy (for example RESOLFT), which requires markers that are capable of enduring multiple cycles of reversible photoactivation (Adam, 2014  ). Controlled phototransformation of PCFPs enables their use in advanced imaging with resolution beyond the diffraction limit of light (Rust et al., 2006  ; Hess et al., 2006  ; Betzig et al., 2006  ); they are excellent markers for localization-based super-resolution microscopy (for example PALM).

Kaede-like FPs (e.g. Kaede, EosFP, DendFP, KikGR, mcavRFP, rfloRFP etc.) form a separate subgroup of irreversible PCFPs with a conserved His62-Tyr63-Gly64 chromophore sequence and characteristic green-to-red photoconversion (PC; Hayashi et al., 2007  ; Mizuno et al., 2003  ; Pakhomov et al., 2004  ; Labas et al., 2002  ; Tsutsui et al., 2009  ; Adam et al., 2009  ; Nienhaus et al., 2005  ). Under UV/blue light (360–410 nm), Kaede-like FPs undergo a green-to-red PC accompanied by a cleavage of the peptide backbone. The absorption spectra of both forms each have two bands at 380/492 nm (green) and 495/555 nm (red) corresponding to the protonated neutral and anionic chromophore, respectively. It has been demonstrated that in Kaede-like proteins only the neutral and not the anionic chromophore is able to photoconvert into the red state. A remarkable exception from this rule is DendFP and its monomeric variant Dendra2, which can be photoconverted in a microscope by continuous-wave blue light at about 490 nm, albeit with a much lower efficiency compared with UV/violet light (Gurskaya et al., 2006  ; Chudakov et al., 2007  ; Makarov et al., 2014  ). A recent extensive wavelength- and pH-dependent study of Dendra2 PC suggested that the green-to-red conversion occurs following the absorption of one photon directly from the excited state of each (neutral and anionic) form of the green protein without intermediate excited states or species requiring the absorption of additional photons (Makarov et al., 2014  ).

Crystallographic studies have revealed a very high similarity between different Kaede-like proteins with respect to the conformation of the chromophore and adjacent residues (Nienhaus et al., 2005  ). Moreover, the chromophore in the green form appears to be ‘pre-adapted’ to PC, as the spatial position of His62 in the green and red forms remains very similar in spite of the protein backbone cleavage and Cα=Cβ double-bond formation. The conformation of the catalytic base Glu211 was found to be equally conserved, with the carboxylate of Glu211 in van der Waals contact with the β-carbon of His62 (3.5 Å; Kim et al., 2015  ). However, the step-by-step detailed chemical mechanisms of light-induced red chromophore formation in Kaede-like proteins remains unknown; several suggested alternative schemes are under debate (Nienhaus et al., 2005  ; Hayashi et al., 2007  ; Tsutsui et al., 2009  ).

Here, we present the results of a crystallographic study of the tetrameric fluorescent protein DendFP (from the soft coral Dendronephthya sp.; subgroup of Kaede-like FPs; Fig. 1  ) in the green form (DendGFP; λexem = 494/506 nm) and the photoconverted red form (DendRFP; λexem = 560/578 nm) at 1.81 and 2.14 Å resolution, respectively. The crystal structure of green Dendra2 (monomeric variant DendFP) has previously been reported by Adam et al. (2009  ), whereas this is the first structure of photoconverted red DendFP to be reported to date. In the present study, particular attention has been paid to the stereochemical features of the DendFP chromophore area, defining its photophysical properties, as well as to the interfaces that are responsible for protein oligomerization. The structural findings were validated by extensive structure-guided site-directed mutagenesis.

Figure 1
Sequence alignment of the Kaede-like proteins. Residues in the nearest environment (within 3.9 Å) of the chromophore (HYG; shown in grey) in DendFP are shown in red (direct hydrogen bonding and hydrophobic interactions) and blue (water-mediated ...

2. Materials and methods  

2.1. Protein preparation and crystallization  

For protein expression, the fragment encoding wild-type DendFP was cloned into pQE-60 vector (providing a C-terminal His tag) and transformed into Escherichia coli JM-109 (DE3). Bacterial cultures were grown overnight at 37°C. The cells were pelleted by centrifugation, resuspended in 20 mM Tris–HCl pH 7.4 buffer containing 100 mM NaCl and lysed by sonication. The recombinant protein was further purified by immobilized metal-affinity chromatography on Ni–NTA resin (Qiagen) according to the manufacturer’s protocol followed by size-exclusion chromatography on a Superdex 200 (16/60) column (GE Healthcare, USA).

The preparation of mutant variants by site-directed mutagenesis was carried out using a QuikChange kit (Stratagene). Absorption and fluorescence spectra of the purified proteins were recorded with Varian Cary 50 UV/Vis and Varian Cary Eclipse fluorescence spectrophotometers. Extinction coefficients for wild-type DendFP and its mutants were determined based on the method of Ward (2006  ) (see Supporting Information for details). The value representing the capability of the mutant to absorb UV light at physiological pH relative to the corresponding value for wild-type (wt) DendFP (RA380) was calculated as

equation image

where A 380 pH 7.5(mut) and A 380 pH 7.5(wt) are the absorbances of mutant and wt DendFP in solution at pH 7.5 and A 380 pH 13(mut) and A 380 pH 13(wt) are the absorbances of denatured mutant and wt DendFP in 0.2 M NaOH solution (pH 13) (see Supporting Information).

A nearly 50% green-to-red PC was achieved after 2 h exposure of 1 mg ml−1 DendFP green protein solution in 20 mM Tris pH 8.0, 200 mM NaCl to 365 nm light (UVLMS-38 EL Series 3UV lamp). The molar extinction coefficients of DendFP reported previously in Pakhomov et al. (2015  ) were used to determine the fractions of green and red forms (Supplementary Fig. S1).

Determination of the PC rate was carried out with protein immobilized on Ni–NTA agarose beads using a Leica DMI6000 inverted fluorescence microscope. PC was triggered by illumination of the beads with 405 nm blue light using the 63× objective with the filter cube for BFP. Increase of red fluorescence was monitored using the filter cube for Texas Red dye. Kinetic curves were approximated with COPASI using a differential evolution algorithm for the estimation of reaction parameters (Hoops et al., 2006  ).

Crystals suitable for X-ray study were grown at 20°C by the hanging-drop vapour-diffusion method. Each drop of DendGFP and DendRFP consisted of 2 µl 20 mM Tris pH 8.0, 200 mM NaCl (protein concentrations of 22.3 and 16.5 mg ml−1, respectively) mixed with an equal amount of reservoir solution consisting of 0.2 M magnesium formate, 20% PEG 3350. Crystals for X-ray data collection reached their final size in two weeks.

2.2. X-ray data collection, structure solution and crystallographic refinement  

X-ray diffraction data were collected from single crystals flash-cooled in a 100 K nitrogen stream. Prior to cooling, the crystals of DendFP were transferred to a cryoprotectant solution consisting of 20% glycerol and 80% reservoir solution. Data were collected with a MAR300 CCD detector on the SER-CAT beamline 22-ID at the Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, USA and were processed with HKL-2000 (Otwinowski & Minor, 1997  ).

Although the data could be successfully merged in P21 and C2221 for DendGFP and DendRFP, respectively, both structures could only be solved in space group P1. The crystal structure of DendFP was solved by the molecular-replacement method with MOLREP (Vagin & Teplyakov, 2010  ; Winn et al., 2011  ) using the coordinates of the crystal structure of mutant Dendra2 (PDB entry 2vzx; Adam et al., 2009  ) as a starting model. Refinement of the structure was performed with REFMAC5 (Murshudov et al., 2011  ), alternating with manual correction of the model with Coot (Emsley et al., 2010  ). The addition of water molecules and structure validation were carried out in Coot. The twin fractions refined by REFMAC5 were 0.51 and 0.49 for DendGFP and 0.28, 0.25, 0.23 and 0.24 for DendRFP, confirming that both structures are perfectly pseudo-merohedrally twinned. Crystallographic data and refinement statistics are given in Table 1  . The coordinates and structure factors of green and red DendFP were deposited in the Protein Data Bank under accession codes 5exb and 5exc, respectively.

Table 1
Crystallographic data and refinement statistics

3. Results  

3.1. Overall structure  

Upon irradiation with UV light (360–400 nm), green DendFP (DendGFP) irreversibly turns into red DendFP (DendRFP) (Fig. 2  a). The absorption and emission spectra of DendGFP are modestly blue-shifted (~10 nm) relative to other Kaede-like proteins. At physiological pH, the absorption spectrum of DendGFP exhibits a major band at 494 nm (with a shoulder at 470 nm) and a minor band at 380 nm, corresponding to anionic (deprotonated) and neutral (proton­ated) chromophores, respectively (Figs. 2  b and 2  c). The corresponding spectrum of DendRFP has bands at 560 nm (with a shoulder at 530 nm) and 445 nm (Figs. 2  b and 2  d), respectively. DendFP demonstrates a significant pH-dependence (Figs. 2  c and 2  d); its absorbance spectra show that below pH 5 the chromophore is almost completely protonated (380/445 nm), whereas above pH 8 the protonated chromophore is hardly detectable. At the physiological pH of 7.5, 82% of the chromophore is deprotonated. The protonated chromophore has a very low quantum yield and barely contributes to the overall brightness of DendGFP (Patterson & Lippincott-Schwartz, 2002  ).

Figure 2
Spectral properties of DendFP. (a) Kinetics of green-to-red photoconversion. (b) Emission spectra of DendGFP and DendRFP. (c, d) pH-dependence of the absorption spectra of DendGFP and DendRFP (the 380 and 445 nm bands correspond to the protonated ...

The principal structural fold of the DendFP monomer, which is shared with all other members of the GFP family, is an 11-stranded β-barrel with loop caps from both sides and a chromophore embedded in the middle of an internal axial helix.

The biological assembly of DendFP in the crystalline state is a tetramer composed of two dimers and is characterized by a 222 point symmetry arrangement. The surface of the subunits in both the green and the red forms of DendFP creates two types of interface: IF1 and IF2 (Fig. 3  ). A relatively weak interface IF1 is located within the dimer between the two antiparallel monomers with a side-to-side packing at ~175° and related by a noncrystallographic twofold symmetry axis. IF1 has a buried contact area of ~840 Å2 (per monomer) and is stabilized by eight symmetry-related hydrogen bonds and a few hydrophobic contacts dispersed over the interface (Table 2  ). Interface IF2 is more extensive than IF1 and has a buried area of ~1600 Å2 (per monomer). IF2 is located between the two monomers of different dimers positioned at ~110° to each other. Interface IF2 is stabilized by six salt bridges, 12 direct hydrogen bonds, a network of water-mediated hydrogen bonds and a number of hydrophobic contacts (Table 2  ). The irregular C-terminal tail beyond Ser218 extends away from the β-barrel towards the cylindrical surface of the adjacent monomer, contributing to the interface stabilization.

Figure 3
Stereoview of the intersubunit interfaces IF1 (a) and IF2 (b) in the DendFP tetramer. This figure was created with PyMOL (DeLano, 2002  ).
Table 2
Polar interactions at DendFP interfaces

The P1 crystal cells of DendGFP and DendRFP contain four and two tetramers, respectively (Table 1  ). The pairwise Cα superposition (residues 3–221) for all monomer combinations in the green, red and green versus red DendFP structures resulted in average r.m.s.d. values of 0.38, 0.55 and 0.49 Å, respectively.

3.2. Green DendFP  

The structure of the chromophore area in DendGFP is nearly identical to that of its monomeric variant Dendra2 reported by Adam et al. (2009  ) and is quite similar to those of other photoconvertible Kaede-like proteins (Nienhaus et al., 2005  ; Hayashi et al., 2007  ; Tsutsui et al., 2009  ). We observed no indication of disorder in the tightly packed chromophore area. The post-translational modification of the chromophore-forming sequence His62-Tyr63-Gly64 in DendGFP results in a coplanar two-ring chromophore consisting of a five-membered imidazolinone heterocycle and a phenolic ring. The geometry of the first chromophore residue, His62, is consistent with that of the ‘green’ chromophore with an sp 3-hybridized Cα atom and the preceding peptide bond adopting a standard trans conformation. The side chain of the second chromophore residue, Tyr63, adopts a cis conformation. In all four tetramers, the phenolic ring of Tyr63 shows a substantial deviation from chromophore planarity, described by torsion angles of 10 to 25° and −20 to −30° around the corresponding Cα—Cβ and Cβ—Cγ bonds. The immediate chromophore environment comprises 21 tightly packed residues (Fig. 4  ) that form three direct and four water-mediated hydrogen bonds to the chromophore. A well-defined water molecule has been found in the proximity of the imidazole ring of His62 in DendFP. This water was suggested to assist the green-to-red PC in Kaede protein (Hayashi et al., 2007  ). The cis form of the chromophore is stabilized by a direct hydrogen bond between the hydroxyl of Tyr63 and the nearby Ser142. These interactions, along with two specific hydrogen bonds, one between the catalytic Arg91 and the imidazolinone carbonyl and the other between Trp89 and Gly64 of the chromophore, as well as π-stacking between the aromatic rings of Tyr63 and the nearby His193, influence the charge distribution over the chromophore and hence its spectral properties. The chromophore immediate environment (within 3.9 Å) in DendFP and other Kaede-like FPs is characterized by high stereochemical homology (Fig. 1  ), with a major difference at positions 105 and 116. Ser105/Gln116 in DendFP and Asn105/Asn116 in Kaede FPs generate stereochemically different local hydrogen-bond clusters, which interact with the chromophore (His62-Tyr63-Gly64) and its flanking residues in positions 61 and 65 in a similar manner (Fig. 5  ). Both Ser105 and Gln116 form a water-mediated hydrogen bond to the same Gly64 carbonyl O atom. Additionally, Gln116 forms direct hydrogen bonds to similar points in Leu61 and Asn65.

Figure 4
The nearest amino-acid environment of the DendGFP chromophore. Hydrogen bonds (within 3.3 Å) are shown as blue dashed lines, water molecules (W) as red spheres and van der Waals contacts (≤3.9 Å) as brown ‘eyelashes’. ...
Figure 5
Local hydrogen-bond clusters interacting with the chromophore moiety in DendGFP (a) and Kaede (b). This figure was created with LIGPLOT/HBPLUS (McDonald & Thornton, 1994  ; Wallace et al., 1995  ).

A direct hydrogen bond between Arg66 and the imidazolinone carbonyl has been shown to be a key structural feature of EosFP, Kaede and KikGR (Nienhaus et al., 2005  ; Hayashi et al., 2007  ; Tsutsui et al., 2009  ; Berardozzi et al., 2016  ). Similar to Dendra2, in DendGFP the side chain of Arg66 does not form a direct hydrogen bond to the imidazolinone carbonyl of the chromophore. Instead, the positive charge of the Arg66 guanidinium group is neutralized by a salt bridge with a negatively charged carboxyl of the catalytic Glu211. This structural difference contributed a ~10 nm blue shift in the fluorescence of DendFP relative to that of other Kaede-like proteins (Adam et al., 2009  ).

3.3. Red DendFP  

DendGFP undergoes an irreversible PC into the red form on irradiation with UV light. This transition results in a 66 and 72 nm bathochromic shift of its absorption and emission bands, respectively. The structure of DendRFP demonstrates the presence of a cleaved protein backbone at Cα—N of the chromophore His62 and a terminal carboxamide group at the preceding Leu61 residue (Figs. 6  and 7  ). The His62 imidazole ring is conjugated with the rest of the chromophore moiety, forming a highly coplanar structure owing to the Cα=Cβ double bond of His62. This double bond provides a trans configuration of the linkage between the imidazolinone and imidazole rings as in other Kaede proteins, except for KikGR, which shows a cis configuration of the linkage (Tsutsui et al., 2009  ). The NH2 group of the Leu61 carboxamide, formed after the photoinduced backbone cleavage, is positioned within a distance of 3 Å from the unprotonated Nδ1 atom of the His62 imidazole.

Figure 6
Structures of the chromophore (His62-Trp63-Gly64) in DendGFP (a) and DendRFP (b). This figure was created with ChemDraw (Cambridge Soft; http://www.cambridgesoft.com).
Figure 7
The electron density (2F oF c, cutoff ρ = 1.0σ) of DendRFP showing the chromophore (His62-Trp63-Gly64) and preceding modified Leu61 with a carboxamide group. This figure was created with PyMOL (DeLano, 2002  ).

It is noteworthy that the stereochemical arrangements in the immediate environment of the chromophore in red and green DendFPs are remarkably similar in spite of the structural difference in the chromophore. The minor distinctions between them were found predominantly around the backbone cleavage site. As in other Kaede proteins, a water molecule found near the imidazole ring of His62 in green DendFP is absent in red DendFP.

3.4. Mutagenesis  

Site-directed mutagenesis was carried out to validate the identity of key amino-acid residues affecting the PC of the DendGFP chromophore (Table 3  ). As in other Kaede-like proteins (Nienhaus et al., 2005  ; Hayashi et al., 2007  ), the Glu211Gln replacement completely abolished green-to-red PC and fluorescence, emphasizing the importance of the conserved Glu211.

Table 3
Spectral and kinetic characteristics of DendFP mutants

Replacement of the chromophore-preceding Leu61 with Gly/Ala and a Leu40Met mutation close to the chromophore His62 affected neither the PC rate nor the spectral properties. On the other hand, Ile195Met and Leu61Ser substitutions caused a moderate increase and decrease in the degree of protonation of the chromophore accompanied by a noticeable acceleration/deceleration of PC, respectively. The red emission of the Ile195Met mutant showed a moderate batho­chromic shift of 9 nm. Leu40Ser, Ile195Ala/Asn and Leu209Ala/Asn replacements yielded nonfluorescent variants, presumably owing to incomplete protein folding.

The Thr59His, Phe173Ala and Ile157Ser variants probing the environment of the phenolic ring of Tyr63 had a moderate influence on its protonation and spectral properties. It was previously noticed that mutating one or more of the residues at positions 157, 159 and 173, located around the chromophore, generally affects the photochromic behaviour of Kaede-like proteins (Adam et al., 2011  ; Ando et al., 2004  ). In the case of DendFP, the Phe173Ala mutant followed this trend, while the Ile157Ser variant did not show any photochromic behaviour. In addition to irreversible green-to-red PC, the Phe173Ala DendFP variant demonstrated a reversible photochromism similar to that reported for Phe173Ser Dendra2 (named NijiFP; Adam et al., 2011  ). On exposure to 405 nm light the Phe173Ala mutant exhibits green fluorescence (ON state), whereas illumination with 488 nm turns its fluorescence OFF, presumably owing to cistrans isomerization of the chromophore (Supplementary Fig. S3).

We hypothesized that an efficient proton donor/acceptor Ser142, hydrogen-bonded to the hydroxyl group of Tyr63, could affect the properties of DendFP. Indeed, the Ser142Cys subsititution resulted in a considerable increase in the protonated fraction of the chromophore and was accompanied by a 4.4-fold acceleration of PC. A Ser142Ala mutation further increased the protonation of the chromophore and showed a 4.6-fold higher PC rate than that of wild-type DendFP. While both the Ser142Cys and Ser142Ala mutants show an impressive acceleration of PC, their fluorescence was much dimmer than that of wild-type DendFP. The absorbance spectra of the Ser142Ala mutant show a dim blue and green emission (455 and 512 nm) that becomes cyan-green and red (491 and 585 nm) after PC, respectively. Unfortunately, the highly protonated Ser142Ala mutant does not permit an accurate characterization of its anionic form.

The imidazole side chain of His193 π-stacking with the chromophore Tyr63 was suggested to stabilize negative charge on its hydroxyphenyl ring. Indeed, the His193Leu variant showed a considerable increase in chromophore protonation, a threefold increase in the PC efficiency and a 5 and 11 nm bathochromic shift of the green and red emission bands, respectively.

Lastly, probing position 116, which is occupied by Asn and Gln in Kaede and DendFP, respectively, found that the Gln116Asn replacement in DendFP did not cause any noticeable change in the spectral properties of the mutant, but significantly suppressed its PC. Introduction of an Ser105Asn mutation in addition to Gln116Asn largely restored the PC, resulting in an increase in its rate by an order of magnitude.

4. Discussion  

DendFPs belongs to the subgroup of photoconvertible Kaede-like FPs, sharing 60–68% pairwise sequence identity and a conservative chromophore-forming sequence His62-Tyr63-Gly64 with them (Fig. 1  ). Under UV/blue light irradiation Kaede-like FPs undergo an irreversible green-to-red PC. This results in the oxidation of the Cα—Cβ bond of His62 accompanied by cleavage of the polypeptide backbone at the Cα—N bond and by the formation of a terminal carboxamide group at the chromophore-preceding Leu61. Conjugation of the chromophore imidazolinone with the imidazole of His62 through the newly created Cα=Cβ double bond extends the π-electron system of the chromophore, providing a 72 nm red shift of the fluorescence (Fig. 2  ). The structures of the chromophore area in the wild-type green DendFP and its monomeric variant Dendra2 reported previously (Adam et al., 2009  ) were found to be nearly identical. Despite a substantial structural difference in the green and red DendFP chromophores, their immediate environment was found to be remarkably similar, with minor distinctions found near the point of backbone cleavage.

Dendra2, a monomeric variant of DendFP, was obtained by eight point mutations (highlighted in Fig. 1  ), including three basic monomerizing replacements: Asn121Lys, Met123Thr and Tyr188Ala (Gurskaya et al., 2006  ). Interface analysis of DendFP shows that only Asn121 at the IF1 interface makes a meaningful contribution to the association of the subunits. Asn121 is part of a six-residue cluster comprising Asn121, Arg119 and Arg104 from the two symmetry-related neighbouring subunits (Fig. 3  a). The pair of Asn121 residues in this cluster form a highly stable inter-subunit hydrogen bond at all IF1 interfaces. The inter-subunit hydrogen bonds between Asn121 and Arg104 or Arg119 show some variability. The replacement of Asn121 by a positively charged Lys disrupts this cluster at IF1, promoting water access to IF2 with the subsequent disruption of its stabilizing contacts, as listed in Table 2  . The mutations Met123Thr and Tyr188Ala at the IF1 and IF2 interfaces, respectively, affect the monomerization only marginally. The first replacement forms destabilizing contacts with the hydrophobic CH3 group of Thr123 and the charged side chains of Glu90 and Arg104 from the interacting subunit. The second replacement removes the stabilizing water-mediated hydrogen bond between the hydroxyl of Tyr188 and the guanidinium group of Arg194 from the interacting subunit. Five other mutations (Fig. 1  ) are located too far from both interfaces to affect monomerization. Positions 40 and 61 reside in the vicinity of the chromophore and could hardly contribute to monomerization. Positions 95, 199 and 213 are close to the surface of the β-barrel; some of them are silent or/and some are presumably required to retain the native fold of the protein.

Stereochemical analysis of the IF2 interface revealed an additional six-residue cluster comprising Asp192, Arg216 and Thr143 of two symmetry-related neighbouring subunits (Fig. 3  b). This cluster stabilizes the oligomeric structure with two salt bridges between Arg216 and Asp192 and two hydrogen bonds between Arg216 and Thr143. Accordingly, an Asp192Arg/Lys replacement, either on its own or in combination with Thr143Ala, could efficiently disrupt the stabilizing cluster further, promoting monomerization.

Structure-based site-directed mutagenesis in the area of the DendGFP chromophore revealed a number of key residues that affect its properties. The efficient proton donor/acceptor Ser142 forms a strong hydrogen bond to Tyr63, stabilizing the anionic form of the chromophore. In all PCFPs reported to date position 142 is occupied by a Ser, which has been suggested to favour a moderate blue shift in the fluorescence of the chromophore (Adam et al., 2009  ). The Ser142Cys and Ser142Ala substitutions decreased the stabilization of the negative charge of Tyr63, resulting in highly protonated variants and a 4.4–4.6-fold acceleration of PC (Table 3  ). Both the Ser142Cys and Ser142Ala variants, with improved PC rates, unfortunately exhibit a dim fluorescence that limits their application in advanced imaging technologies.

The imidazole side chain of His193 participates in efficient π-stacking with the phenolic ring of Tyr63. In cyan amFP486, the histidine forming analogous π-stacking was suggested to carry a positive charge, stabilizing the charge on the hydroxyphenyl ring of the chromophore, and to favour a blue shift in the fluorescence (Henderson & Remington, 2005  ). In DendFP, a His193Leu substitution abolished this stabilization, causing a threefold increase in the PC efficiency and a 5 and 11 nm bathochromic shift of the green and red emission bands, respectively (Table 3  and Fig. 8  ). These observations are in a good agreement with a blue shift of Thr59His variant fluorescence, where the second π-stacking of His59 with the phenolic ring of Tyr63 provides an additional stabilization of the anionic chromophore.

Figure 8
Correlation between the PC rate and the relative capability of the mutant to absorb UV light at physiological pH (RA380). The point numerical markings on the graph correspond to the number given to each mutant as in Table 3  .

Another important feature of DendFP that is worth mentioning is the absence of a direct hydrogen bond between the side chain of Arg66 and the imidazolinone carbonyl as observed in other Kaede-like proteins (Kaede, EosFP, KikGR etc.). Previously, the absence of this hydrogen bond has been suggested to decrease the stabilization of the imidazolinone negative-charge distribution, which contributed to a ~10 nm blue shift of the fluorescence (Adam et al., 2009  ). Instead, in DendFP Arg66 neutralizes its positive charge by making a salt bridge with the catalytic Glu211. A recent study by Berardozzi et al. (2016  ) showed that the observed conformation of Arg66 in DendFPs is stabilized by hydrogen bonding between the [sm epsilon]-amine of the Arg66 guanidinium moiety and the nearby hydroxyl of Thr69, which is located 8 Å away from the chromophore (versus Ala69 in other Kaede-like proteins). It was demonstrated that replacement of Thr69 with Ala in Dendra2 and, vice versa, Ala69 with Thr in Kaede-like mEos2 switches the conformations of the side chains of Arg66 and completely swaps the photophysical behaviour of the two FPs.

DendFP and other Kaede-like proteins are characterized by high amino-acid homology of the immediate environment of the chromophore (within 3.9 Å); their second most important difference is at positions 116 and 105, which are occupied by the adjacent Gln and Ser in DendFP and two Asn residues in the other Kaede-like proteins (Fig. 1  ). In DendFP, both Ser105 and Gln116 form a water-mediated hydrogen bond to the carbonyl O atom of the chromophore Gly64, while Gln116 also forms direct hydrogen bonds to the carbonyl O atom of Leu61 preceding the chromophore and Asn65 following the chromophore (Fig. 5  ). In Kaede, Asn105 forms a direct hydrogen bond to the same carbonyl O atom of Gly64, whereas Asn116 forms water-mediated hydrogen bonds to similar points of the Phe61 carbonyl O atom and Asn65. A series of mutations have shown that in DendFP, Asn in position 105 or in both positions 105 and 116 (as in Kaede) significantly decreases the PC rate. Moreover, a single Gln116Asn replacement leads to an unprecedented 30-fold decrease in the PC rate (Table 3  ), presumably owing to perturbation of the local hydrogen-bonding network of the chromophore caused by the shorter side chain of Asn. Introduction of the second mutation, Ser105Asn, largely restores the PC, resulting in an increase in the PC rate by an order of magnitude.

While only the neutral and not the anionic chromophore in Kaede-like proteins possesses the capacity to photoconvert to the red state, Dendra (and its monomeric variant Dendra2) can be photoconverted from both neutral and anionic forms. The PC quantum yields of Dendra2 suggest a 25-fold more efficient green-to-red conversion when initiated in the neutral form (Makarov et al., 2014  ). In numerous instances it has been noted that there is no simple one-to-one correlation between the pK a of the chromophore hydroxyphenyl group and the PC efficiency (Moeyaert et al., 2014  ). However, here we have observed a good correlation between the chromophore protonation and its PC rate, with the exception of position 142. We plotted the PC rate against RA380, the value representing the relative capability of the mutant to absorb UV light at physiological pH (see §2 and Supporting Information for details; Fig. 8  ).

The PC rate of the Ser142Cys and Ser142Ala variants noticeably deviates from the linear trend followed by the other mutants. Such deviation is presumably owing to the increased flexibility of the chromophore phenolic ring caused by weakening/disruption of the hydrogen bond to the residue at position 142. These data are in line with the previously proposed mechanism, in which the PC is triggered by excited-state chromophore motions promoted by a decreased density of molecular packing in the immediate chromophore environment (Kim et al., 2013  ). However, such an increase in the chromophore mobility often decreases its quantum yield, drastically impacting the usability of the corresponding mutants (Mandal et al., 2004  )

5. Conclusions  

Here, we have presented the X-ray structures of wild-type DendFP in green and photoconverted red forms. Under UV/blue light irradiation DendFP undergoes an irreversible green-to-red PC, resulting in the formation of a double Cα=Cβ bond in His62 of the chromophore and cleavage of the peptide backbone at the Cα—N bond. The following combination of key structural features was shown to be important for the fluorescence and PC of DendFP.

  • (i) The absence of direct hydrogen bonding between Arg66 and the chromophore imidazolinone is the critical structural feature that distinguishes DendFP from other red-shifted Kaede proteins in which this hydrogen bond is present.
  • (ii) Replacement of the efficient proton donor/acceptor Ser142 with the less potent Cys/Ala or the removal of the π-stacking interaction between His193 and the chromophore Tyr63 by the replacement of His193 with Leu increases the PC rate and causes a red shift of the fluorescence.
  • (iii) The local hydrogen-bond network comprising the Ser105 and Gln116 pair and the chromophore with flanking residues greatly affects the PC rate.

Supplementary Material

PDB reference: green fluorescent protein DendGFP, 5exb

PDB reference: red fluorescent protein DendRFP, 5exc

Supporting Information.. DOI: 10.1107/S205979831601038X/mn5111sup1.pdf

Acknowledgments

This research was supported by funding from the Russian Science Foundation (project 14-14-00281). X-ray data collection at the synchrotron station was supported in part by Federal funds from the National Cancer Institute, National Institutes of Health under contract No. HHSN261200800001E and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Diffraction experiments were carried out on synchrotron beamline 22-ID of the Southeast Regional Collaborative Access Team (SER-CAT) located at the Advanced Photon Source, Argonne National Laboratory. Use of the APS was supported by the US Department of Energy, Office of Science and Office of Basic Energy Sciences under Contract No.W-31-109-Eng-38. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the US Government.

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