Yellow and Orange Fluorescent Proteins with Tryptophan-based

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Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores Nina G. Bozhanova,*,†,§ Mikhail S. Baranov,†,‡,§ Karen S. Sarkisyan,† Roman Gritcenko,⊥ Konstantin S. Mineev,†,∥ Svetlana V. Golodukhina,† Nadezhda S. Baleeva,†,‡ Konstantin A. Lukyanov,† and Alexander S. Mishin† †

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia Pirogov Russian National Research Medical University, Ostrovitianov 1, 117997 Moscow, Russia ⊥ Centre for Analysis and Synthesis, Department of Chemistry, Lund University, 22100 Lund, Sweden ∥ Moscow Institute of Physics and Technology, Institutsky per., 9, 141701 Dolgoprudny, Russia ‡

S Supporting Information *

ABSTRACT: Rapid development of new microscopy techniques exposed the need for genetically encoded fluorescent tags with special properties. Recent works demonstrated the potential of fluorescent proteins with tryptophan-based chromophores. We applied rational design and random mutagenesis to the monomeric red fluorescent protein FusionRed and found two groups of mutants carrying a tryptophan-based chromophore: with yellow (535 nm) or orange (565 nm) emission. On the basis of the properties of proteins, a model synthetic chromophore, and a computational modeling, we concluded that the presence of a ketone-containing chromophore in different isomeric forms can explain the observed yellow and orange phenotypes.

F

designing improved variants are hampered by the lack of understanding of chromophore structure. Chemical synthesis of chromophore analogs, a useful approach to understanding the properties of protein chromophores, was successfully applied to a number of proteins with tyrosine-containing chromophores11−14 and tryptophan-based cyan fluorescent proteins4 but was never used to aid the understanding of spectral properties of yellowand orange-emitting proteins with Trp66. Here, we explored the potential of monomeric fluorescent protein FusionRed15 to accommodate a tryptophan-based chromophore, developing its yellow and orange mutants. On the basis of the analysis of properties of the mutants, we proposed a keto group containing chromophore structure for these red-shifted Trp66-containing fluorescent proteins. Synthesis of the model compound and theoretical calculation allowed us to reveal the high impact of the keto group containing chromophore geometry on its spectra.

luorescent proteins are essential instruments of modern fluorescence microscopy. Spectral properties of fluorescent proteins are determined mainly by the structure of the autocatalytically formed aromatic compound, the chromophore, but also can be substantially affected by the state of chromophore ionization, isomerism, and its interactions with the amino acid surroundings. The chromophore of the first discovered fluorescent protein, green fluorescent protein (GFP), forms by intramolecular condensation and oxidation of three amino acids (Ser65− Tyr66−Gly67; here and below, we use numbering according to Aequorea victoria GFP, see SI, Figure S1). Amino acid at position 66 essentially defines the spectral properties of a chromophore. For unclear reasons, all naturally occurring fluorescent proteins carry tyrosine residue at position 66. However, its substitution to other aromatic amino acids (Phe, Trp, His) is also compatible with the ability to fluoresce in the visible spectrum1 and led to the development of a wide spectral range of fluorescent tags.2 In particular, Tyr66Trp substitution has led to the development of the cyan protein with 93% quantum yield,3 green protein with the longest fluorescence lifetime,4,5 and orange genetically encoded photosensitizer.6 Moreover, proteins with tryptophan-based chromophores turned out to be excellent donors for FRET-based applications, with cyan−yellow pairs being currently the most used FRET pair.7,8 However, only a few such tags were developed that fluoresce in the yellow and orange regions of the spectrum.6,9,10 The rational approaches to © 2017 American Chemical Society



RESULTS AND DISCUSSION Mutagenesis of FusionRed. We performed site-directed mutagenesis introducing substitution Y66W into the FusionRed coding sequence. Expression of the mutant gene (FR-Y66W) in E. coli resulted in a very weak fluorescence of colonies,

Received: April 21, 2017 Accepted: May 19, 2017 Published: May 19, 2017 1867

DOI: 10.1021/acschembio.7b00337 ACS Chem. Biol. 2017, 12, 1867−1873

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Table 1. Optical Properties of the FusionRed Mutant Variants in Comparison with Previously Published TryptophanContaining Orange Proteinsa

a

mutations additional to Y66W

excitation, nm

emission, nm

Stokes shift, nm

FQY, %

EC, M−1·cm−1

maturation half-time, h

M14T, N192K (FR66W-Yellow) C165S 165A C165G M14T, K15R, C165G (FR66W-Orange) emKate_Y64W9 mHoneydew10

490 499 490 529 530 479 487/504

535 543 553 565 565 558 537/562

45 44 63 36 35 79 50/58

28 n.d. 25 n.d. 26 n.d. 12

22000 n.d. 29000 n.d. 19000 n.d. 17000

8.5 n.d. 11.5 n.d. 10.5 n.d. n.d.

n.d.: not determined. FQY: fluorescence quantum yield. EC: extinction coefficient.

developed gradually over the course of a five-day incubation of plates at 4 °C. To find compensatory substitutions that rescue fluorescence of FR-Y66W, we used random mutagenesis. Interestingly, it resulted in two groups of mutants possessing either yellow or orange fluorescence. The brightest variants from each group were proteins FR66W-Orange (FusionRed M14T/K15R/Y66W/C165G) and FR66W-Yellow (FusionRed M14T/Y66W/N192K; Table 1, Figure 1, and SI Figure S1). Preservation of the monomeric state of FusionRed by these mutants was confirmed by size-exclusion chromatography (SI, Figure S2).

We also performed random mutagenesis of the amino acids near the chromophore of the FusionRed-Y66W (148, 165, and 203 positions). Most of the colonies were nonfluorescent after 20 h at 37 °C except the mutants FR66W−C165S and FR66W−C165A with prominent yellow fluorescence (Table 1). Amino acid position 165 is placed close to the chromophore (SI, Figure S3) and is well-known to be important for the fluorescent protein properties, including protonation state, cis− trans isomerization, and polarization.2 Here, we see a clear influence of this position on spectra of FR66W mutants: the smallest residue (Gly165) leads to the orange phenotype, whereas bigger side chains (in a row of Ala, Ser, Cys) result in a gradual blue shift of emission as well as a sharp 30−40 nm blue shift of excitation maxima (Table 1). The role of position 192 is less clear. According to the X-ray structure of the related protein mKate,16 amino acid 192 is placed in the loop and has no direct interaction with the chromophore (SI, Figure S3). However, it could influence on the chromophore surroundings through slight repositioning of the 9th and/or 10th β-sheets. Despite the low brightness and comparatively slow maturation (Table 1 and SI, Figure S4) of the proteins, mammalian cells expressing FR66W-Orange, FR66W-Yellow, and FR66W−C165A can be imaged under the moderate light intensities allowing acquisition of well-resolved pictures in widefield and confocal modes (SI, Figure S5) and showing photostability similar to eYFP or even higher (SI, Figure S6). Unlike the wide and double-peaked emission curve of mHoneydew,10 all obtained mutants fall into two spectral groups: orange and yellow. We sought to decipher the nature of these spectral groups by characterizing their representative members: FR66W-Yellow and FR66W-Orange. Chromophore. According to the published data for fluorescent proteins,14,17−21 the spectral diversity can be explained by formation of distinct chromophores and/or their presence in different states. The following process of the chromophore formation for FR66W-Yellow and FR66W-Orange can be proposed (Scheme 1, path A): imidazolone ring closure along with an additional oxidation of the Cα−N bond of a residue at position 65 result in the appearance of an acylimine (DsRed-like) chromophore. The further hydrolysis can lead to the backbone cleavage and the formation of the chromophore with a keto group (asFP595like14), which is expected to be more red-shifted than the acylimine-containing (DsRed-like).14,17,18,22 A competing maturation pathway (Scheme 1, path B) can lead to a GFP-like chromophore, manifesting itself as an additional blue-shifted absorption maximum (Figure 1 and SI, Figure S7).

Figure 1. Optical spectra of FR66W-Orange (top) and FR66W-Yellow (bottom). Black solid line, absorbance; dashed colored line, excitation; solid colored line, emission.

To clarify the role of the discovered random substitutions, we reintroduced these mutations one-by-one. Only the proteins FR66W−C165G and FR66W−N192K showed a clearly detectable orange and yellow fluorescence, respectively. FR66W−C165G was almost as bright as FR66W-Orange, while FR66W−N192K was dimmer than FR66W-Yellow (data not shown). 1868

DOI: 10.1021/acschembio.7b00337 ACS Chem. Biol. 2017, 12, 1867−1873

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ACS Chemical Biology Scheme 1. Proposed Chromophore Formation Pathways

tion and spectral behavior under different pH conditions and upon denaturation, as well as by comparison of spectral properties of FusionRed variants with the properties of chromophores’ synthetic analog. Proteins. Behavior upon Denaturation. Protein denaturation exposes groups of the chromophore otherwise buried within the protein. Subsequent gel electrophoresis analysis gives information about protein chain continuity. Together these data may point out the state of the chromophore in the native protein. We assessed FR66W-Yellow and FR66W-Orange using standard SDS-PAGE with or without preliminary boiling (Figure 2). All samples showed three bands with an apparent

Besides formation of distinct chromophores (asFP595-like and DsRed-like), there are a number of other processes which can lead to the red-shifted spectra of the proteins. Deprotonated chromophores within fluorescent proteins are usually significantly red-shifted in comparison to neutral ones.2 This stands for the tryptophan-based chromophores, too (Scheme 2), albeit there are difficulties in stabilizing the anionic state within the protein4,5 due to the very high pKa of the indole fragment. Scheme 2. Possible Isomerism and Ionization of the Tryptophan-based Chromophore

Figure 2. SDS-PAGE analysis of the proteins on 15% gel. (1, 2, 3) FR66W-Yellow (the unboiled and boiled over 0.5 or 10 min samples, respectively). (4) Molecular weight standards. (5, 6, 7) FR66WOrange (the boiled over 10 or 0.5 min and unboiled samples, respectively).

The cis−trans isomerization of tyrosine-based chromophores is also typically accompanied by spectral shifts. Fluorescent proteins with tryptophan-based chromophores could carry multiple isomers: E, Z-trans and Z-cis (Scheme 2). Z-cis−Ztrans isomerism is thought to be responsible for reversible photoconversion of green tryptophan-based fluorescent protein NowGFP.5 Additionally, the process of Z/E photoisomerization was investigated on the model chromophores (including CFP and GFP).23 It was shown that the absorbance spectra of these isomers of the model compounds are not equal in several cases. We addressed the possible roles of all these mechanisms in FusionRed variants by analyzing protein backbone fragmenta-

molecular mass of 27, 20, and 8 kDa. We attributed the 27 kDa to the full-size protein molecule. Two small bands (8 and 20 kDa) appeared to be the result of the intramolecular cleavage of the protein chain near the chromophore. The weak 22 kDa band in the unboiled samples probably represented nondenatured or partly denatured protein. Importantly, there was no significant difference between the intensity of the small bands in the boiled (0.5 and 10 min) and unboiled samples. Therefore, the intramolecular cleavage of the 1869

DOI: 10.1021/acschembio.7b00337 ACS Chem. Biol. 2017, 12, 1867−1873

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ACS Chemical Biology chain was either present in the native protein or occurred very easily. The full-length protein band (27 kDa) likely corresponds to protein species with an immature chromophore. We then investigated spectra changes upon protein denaturation. The presence of 1% SDS in solution resulted in fluorescence loss and formation of a single band in absorption spectra for both FR66W-Orange and FR66W-Yellow (SI, Figure S8). The resulting absorption maxima were very close to that of native FR66W-Yellow and likely arose from the absorption of the chromophore in keto form (asFP595-like, Scheme 1) because the acylimine is not stable under these conditions. Thus, we concluded that the chromophore of FR66W-Yellow was present in the keto form. Though the absorption spectrum shift of FR66W-Orange upon denaturation could be explained by degradation of the acylimine, the presence of acylimine chromophore in FR66WOrange is also unlikely, since it was shown for tyrosinecontaining proteins that acylimine chromophores are blueshifted compared to the keto chromophores.17 Hence, FR66WOrange may carry a different isomer or protonated state of the keto chromophore (Scheme 2). Effect of pH on Spectral Properties of Proteins. Fluorescent proteins with the ionized chromophores are typically very sensitive to changes in pH due to either direct chromophore titration or titration of amino acid side chains involved in chromophore stabilization. This behavior is reported for both tyrosine-based2 and tryptophan-based4,5 fluorescent proteins and can even be deliberately exploited for the development of various pH-sensitive fluorescent indicators.2 To check whether the difference in spectra of orange and yellow proteins could be explained by the protonation state of the chromophore, we assessed their behavior at various pH’s. The proteins showed robust fluorescence in a wide range of pH values (SI, Figures S9−S11). FR66W-Orange exhibited bright orange fluorescence from pH 4.9 to 11.0. The intensity of FR66W-Yellow fluorescence dropped less than 20% in pH range from 6.5 to 11.0. Both further acidification and alkylation of the solution led to a significant fluorescence decrease, but without any spectral shifts. Thus, we concluded that the formation of the anionic form in the orange group seems to be improbable. Photoswitchable Behavior of FR66W-Yellow. In fluorescent proteins with a tyrosine-based chromophore, only one of the possible isomers (cis- or trans-) is usually stabilized by the surrounding amino acids, as evident from multiple crystal structures.24 However, the switch between the isomers is sometimes possible and could be induced by pH change16 or illumination.25 We tried to induce the switching between isomers by irradiating bacteria expressing FR66W-Orange or FR66WYellow with intense light of different colors. The photoswitchable behavior was found for FR66W-Yellow. Short irradiation with blue light led to the decrease of the fluorescence intensity in the green channel and its increase in red (Figure 3). After ∼30 min, the initial fluorescence was restored. The reversibility of this process once again confirms the presence of a similar chromophore structure in both proteins, since the ketone to acylimine transformation is irreversible. Model Compound. We sought to further test the hypothesis of the chromophore structure by investigating the properties of its synthetic analogue. Therefore, we synthesized the model compound 1, mimicking the proposed tryptophan-

Figure 3. Photoswitchable behavior of FR66W-Yellow. (A) Fluorescence emission spectra of E. coli cells containing FR66WYellow protein measured using a Leica confocal microscope. Blue line, before the irradiation; red line, after 7 s of irradiation with blue light (488 nm excitation laser line, 100 μW); black line, 10 min after the irradiation. (B) Bacterial streak of E. coli containing FR66W-Yellow protein before and after 5 min of irradiation with blue light (460−490 nm, 14 mW/cm2) in the green (1) and red (2) channels.

Scheme 3. Synthesis of the Model Compound 1 (asFP595like Chromophore from Scheme 1)

based keto chromophore of FR66W-Yellow and FR66WOrange (Scheme 3). The double bond in the chromophore structure defines the existence of Z and E isomers (Schemes 2 and 3). The Z isomer was found to be more stable: if the E form (or mixture) was obtained in synthesis, the slow transformation into pure Z form in solutions was typically observed.26 The NMR spectra of the model compound 1 showed prevalence of the Z form (93+%, 1-Z, presented in Scheme 3). However, in the presence of the strong acid, the solution of the compound 1-Z transforms into the mixture enriched (up to 75%) with the E form (1-E, Scheme 3). In neutral pH, it transforms back to the 1-Z form rather rapidly (it takes less 1870

DOI: 10.1021/acschembio.7b00337 ACS Chem. Biol. 2017, 12, 1867−1873

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Figure 4. NMR spectra of the model compound 1. (A) Fragments of 1H NMR spectra of 1-E enriched mixture, acquired within 24 h at 30 °C, starting from the sample containing 70% E-isomer (magenta). Assignment of Z- and E-isomer protons is shown. (B) Two stable conformers, 1-Ztrans and 1-Z-cis, and the fragments of 13C NMR spectra of 1-Z, acquired at 30 (red) and 60 °C (blue). Peaks that were broadened due to the hindered rotation of the indole ring are indicated by asterisks.

the amino-acid surroundings, both forms of the model compound 1 possess the absorbance spectra close to that of the FR66W-Yellow, and the nature of the FR66W-Orange chromophore remains unclear (Figure 5). Theoretical Study of Optical Properties. Some of the signals in NMR spectra of the model compound 1-Z were broadened. This effect was especially significant in the 13C spectra where heating of the sample was necessary to resolve all the signals (Figure 4b). We hypothesized that this broadening is the manifestation of dynamic processes in our model system. Most likely, the rotation of the indolyl part around a single C− C bond in the model compound 1-Z that leads to two stable conformers1-Z-trans and 1-Z-cisis responsible for that (Figure 4b). In order to support this hypothesis, we performed calculations of thermochemical and excited state properties of the system using ORCA 3.0.3 software.27 Geometry optimizations as well as frequency calculations were performed at the RI-TPSS-D3BJ/Def2-TZVP level of theory in a vacuum. To increase accuracy, single point energies were obtained at RIJCOSX-SCS-MP228/Def2-QZVP; solvent (MeCN) was taken into account by means of the COSMO solvation model (SI, Figures S16−S19 and corresponding text). Calculations showed that the following three species mainly exist in the solution: 1-Z-trans (dominant form, 78.6%), 1-Z-cis (less stable, 15.7%), and 1-E-trans (5.7%, well in the range of 2−6% estimated by NMR). Isomerization barriers between 1-Z-trans and 1-Z-cis were predicted to be feasible at RT (SI, Figure S20). Therefore, the above-mentioned results suggest that the absorption spectra of the model compound 1-Z likely represent properties of mixtures of various conformers. Next, we performed additional computational studies (including various single-reference methods, for instance, TDDFT and ab initio CIS(D)) to investigate the influence of chromophore geometry on its absorption properties. As can be clearly seen in Figure 6a, calculations showed high dependence of the S0 → S1 transition energy as well the oscillator strengths on dihedral angle. The nature of transitions on selected conformers is depicted in Figure 6c. The 1-Z-trans isomer shows a red shift in absorption spectra in comparison to its cis-

than an hour in alcohols and 3−4 h in DMSO) but could be fixed in the solid state (see procedures in SI). The formation and disappearance of the E isomer were clearly visible in the 1H NMR spectra, and the positions of the signals were in good agreement with the proposed structures (Figure 4a and SI, Table S1). Similarly to other fluorescent protein chromophores, synthetic chromophores showed a very low fluorescence quantum yield (less than 10−5); therefore, only the absorption spectra of the compounds were studied. The absorbance titration of the model compound 1 demonstrates that the indolyl part of the molecule can be deprotonated (SI, Table S2, Figure S13) and characterized with pKa 10.5 in water (SI, Figures S14 and S15). However, the positions of absorbance maxima of anionic form did not show a good correlation with the proteins’ maxima, which additionally confirm the claim about the presence of a neutral form of the chromophore in both groups. However, we also observed no significant difference in the absorbance maxima of the neutral forms of 1-Z and 1-E (Figure 5 and SI, Table S2). In the polar solvents, which better mimic

Figure 5. Absorption spectra of 1-Z (solid red line) and 1-E enriched mixture (solid blue line) in ethanol in comparison to the spectra of FR66W-Yellow (dashed green line) and FR66W-Orange (dashed orange line). 1871

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Figure 6. Influence of geometry on excitation energies of model compound 1-Z. (A) Oscillator strength and wavelength of absorption maxima dependencies toward the dihedral angle between two parts of a conjugated π system at the TD-B2PLYP/Def2-TZVP(-f) (COSMO:MeCN) level of theory. (B) Comparison of calculated absorptions obtained at the TD-B2PLYP/Def2-QZVP level of theory, their Boltzmann weighted sum, and experimental spectrum of 1-Z (in MeCN). (C) S0/S1 difference densities from CIS(D)/Def2-QZVP (COSMO:MeCN) calculations; blue is increasing of electron density upon excitation; red, decreasing.

isomer (1-Z-cis) and E-isomer (Figure 6a,b). This result is further supported by different methods (SI, Figure S22). The 1-Z-twist isomer shows significant red shift, but this is an artifact of mistreatment of the charge-transfer (CT) type transition by DFT based methods and confirmed by CIS(D) results (SI, Figure S21); additionally, the corresponding oscillator strength is nearly zero. In order to evaluate theoretically obtained results, we compared the sum of Boltzmann population weighted (as described in the paragraph above) S0 → S1 absorptions of the most stable conformers with the experimental spectrum of model compound 1-Z. As can be seen in Figure 6b, it matches the experiment quite well. Theoretical studies confirmed a noticeable impact of chromophore geometry on its S0 → S1 absorptions and give a rational explanation for differences in absorption spectra of the studied proteins. Moreover, not only stable conformers have different absorption maxima (Figure 6b) but also intermediate conformers, where the dihedral angle between indole and imidazolone moieties lies between 0° and 180°, have different excitation energies as well as oscillator strengths (Figure 6a, and SI, Figures S22 and S23). Such intermediate conformers have an even greater effect on absorption maxima than stable ones.

that two of them, the ones arising from indolyl part asymmetry, transform to each other easily. While this equilibrium takes place in solutions of the model compound, the particular isomer could be stabilized within the protein environment. This explains the discrepancy of the absorbance spectra between model tryptophan-based chromophores and corresponding proteins that was observed in this work for red-shifted proteins and previously for CFP. Computational studies show a noticeable red shift in absorption spectra of one of the isomers (Z-trans). Thus, we concluded that this isomer is likely characteristic for the orange proteins, while the yellow proteins can contain any other (Z-cis or E). This assumption is also in agreement with the results of site-directed mutagenesis. Indeed, small amino acids at position 165 (Figure S3) in orange proteins provide additional space required for the Z-trans chromophore.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00337. Materials and methods, protein sequences, size-exclusion chromatography, kinetics of maturation, widefield and confocal microscopy, photobleaching data, synthetic procedures, compounds characterization, titration curves, spectroscopic data, and theoretical calculations of the model compound properties (PDF)



CONCLUSIONS We presented two novel groups of the proteins with yellow (535 nm) or orange (565 nm) fluorescence based on the monomeric red fluorescent protein FusionRed. To the best of our knowledge, it is only the fourth example of the red-shifted tryptophan-containing proteins and the first one with the tolerance in a wide range of pH’s. The protein’s behavior upon denaturation and pH titration allows us to claim the keto structure of the chromophore instead of acylimine, previously suggested for similar proteins. The structure of the tryptophan-based keto chromophore implies the formation of three stable isomers. We demonstrated



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Nina G. Bozhanova: 0000-0002-2164-5698 1872

DOI: 10.1021/acschembio.7b00337 ACS Chem. Biol. 2017, 12, 1867−1873

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§

These authors contributed equally.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank A. Kotlobay for the assistance with HPLC. This work was supported by the grant MK-7495.2016.4 of the President of Russian Federation and Molecular and Cell Biology Program of Russian Academy of Sciences. The chemical part of the reported study was funded by the Russian Foundation for Basic Research, according to the research project No. 16-33-60116 mol_a_dk. This research was partially carried out using the equipment provided by IBCH core facility (CKP IBCH).



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DOI: 10.1021/acschembio.7b00337 ACS Chem. Biol. 2017, 12, 1867−1873