ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 3, pp. 340–343. © Pleiades Publishing, Ltd., 2017. Original Russian Text © A.A. Pakhomov, R.V. Chertkova, I.E. Deyev, A.G. Petrenko, V.I. Martynov, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 3, pp. 326–329.
LETTER TO THE EDITOR
Generation of Photoactivatable Fluorescent Protein from Photoconvertible Ancestor A. A. Pakhomov1, R. V. Chertkova, I. E. Deyev, A. G. Petrenko, and V. I. Martynov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia Received November 7, 2016; in final form, November 11, 2016
Abstract⎯The DendFP protein from Dendronephthya sp. converts from the green to red fluorescent state under UV light. We have obtained the mutant variant of the protein, which, in contrast to original DendFP, tends to be phototransformed from the nonfluorescent form to the green fluorescent state. Keywords: GFP, fluorescence, fluorophore, photoactivatable proteins, photoswitchable proteins, photoconvertible proteins DOI: 10.1134/S106816201703013X
INTRODUCTION Fluorescent proteins (FP) of the GFP family are genetically encoded fluorophores, which are used for visualization of processes in living cells [1–4]. A special FP group includes phototransformable proteins (PTFP), which can change their photophysical properties in response to irradiation with light of a specific wavelength [5]. The interest to PTFP is largely stimulated by their use in subdiffraction microscopy, which allows one to visualize cellular objects with a resolution in the nanometer range [6]. There are currently three subgroups of phototransformed FPs. The first one includes photoactivatable proteins, which are irreversibly transformed from the nonfluorescent to the fluorescent state under light irradiation [7]. The second subgroup comprises photochromic proteins, the fluorescence of which is reversibly switched on and off under irradiation with light of two different wavelengths (photoswitchable proteins) [8, 9]. The third subgroup includes photoconvertible proteins, which irreversibly change the fluorescence color under light irradiation. The proteins of the Kaede subfamily (Kaede is the first protein of this subfamily from Trachyphyllia geoffroyi [10, 11]) are subjected to this irreversible transformation from the green to red fluorescent state. Along with Kaede, this subfamily contains DendFP [12–14], 1 Corresponding
author: phone: +7 (495) 336-51-11; fax: +7 (495) 336-61-66; e-mail:
[email protected]. Abbreviations: DendFP, fluorescent protein from Dendronephthya sp.; EosFP, fluorescent protein from Lobophyllia hemprichii; GFP, green fluorescent protein from Aequorea victoria; Kaede, fluorescent protein from Trachyphyllia geoffroyi; KikGR, mutant green fluorescent protein from coral Favia favus; mMaple, mutant fluorescent protein from Clavularia sp.; PAFP, photoactivatable fluorescent proteins; FP, fluorescent proteins; PSFP, phototransformed fluorescent proteins.
EosFP [15, 16], KikGR [17, 18], mClavGR [19], mMaple [20], and other proteins. In this work, the photoconvertible DendFP protein, which changes its fluorescence from green to the red one under light irradiation, was transformed by mutagenesis to the photoactivatable protein, which acquired the green fluorescence under UV irradiation. RESULTS AND DISCUSSION Photoconversion of the DendFP protein from the green to red fluorescence state under UV light is due to the elimination of the amide group in the vicinity of the chromophore, which leads to the expansion of the chromophore π-system due to the insertion of the imidazole ethylenyl group (Fig. 1a) [13]. The photoconversion rate was shown to significantly depend on the state of the chromophore phenolic group. The more the equilibrium is shifted toward the protonated form, the faster the reaction, because the protonated chromophore is capable of absorbing UV light [14]. It should be noted that DendFP containing the protonated chromophore has almost no fluorescence before the phototransformation because the absorbed light energy is consumed mainly in the elimination reaction. After the phototransformation, however, the protein containing the protonated chromophore can fluoresce in the green spectral range with maxima at 501 and 536 nm [21]. Due to this property, DendFP significantly differs from other fluorescent proteins containing the protonated chromophore, which either does not fluoresce or is subjected to the proton transfer in the excited state [22]. The amino acid environment of the chromophore has a significant effect on the proton equilibration. According to X-ray data, the Ser142 residue in
340
GENERATION OF PHOTOACTIVATABLE FLUORESCENT PROTEIN
(а)
(b)
HO
HO
O
N
O
N
hυ
NH O
O
N
Photoelimination
(NRV...)
+ N
(LAT...)
O
N
NH N
341
NH N
CRO
(NRV...) NH2
O (LAT...)
Ser142
Green fluorescent form
Nonfluorescent form
Fig. 1. Photoconversion of the DendFP chromophore. The chromophore in the parent DendFP protein exists in the anionic form; photoconversion is accompanied by the transfer from the green fluorescent to red form. The chromophore in the mutant DendFP-S142A variant is in the protonated (phenol) form and does not fluoresce in the initial state; after photoreaction, it is converted to the green fluorescent form (a); the amino acid environment of the chromophore (CRO) in DendFP (PDB ID 5EXB); dotted lines designate hydrogen bonds between the chromophore and its environment (b).
cence emission peak was at 501 nm, i.e., this protein became green fluorescent. For the comparison, the wild-type protein after the UV irradiation has red fluorescence with an absorption maximum at 560 nm and a maximum of fluorescence emission at 578 nm. Unlike the wild-type protein, the DendFP-S142A mutant was not transformed from the green to red fluorescence state (Fig. 2d). The predominant formation of the green fluorescent form of the protein was observed in this case (Fig. 2c). Thus, the introduction of the Ser142Ala mutation in the photoconvertible DendFP protein led to the shift in the proton equilibrium to the protonated form of the chromophore both before and after the light irradiation. As a result, DendFP-S142A acquired the properties of the photoactivatable protein, i.e., this protein was irreversibly converted from the colorless and almost nonfluorescent state to the f luorescent form.
DendFP is particularly important. It forms the hydrogen bond with the phenol group of the chromophore (Fig. 1b). Site-directed mutagenesis allowed us to obtain the DendFP variants with the replacement of Ser142 by Thr, Cys, and Ala. These changes actually led to a shift in the proton equilibrium toward the protonated chromophore. According to absorption spectra, the proportion of the phenol (protonated) form of the chromophore in the wild-type protein at pH 7.5 was 18% compared to the phenolate (anionic) form. In the mutant variants, this proportion was 50, 74, and 94% for DendFP-S142T, S142C, and S142A forms, respectively. Thus, the chromophore was almost completely protonated in the DendFP-S142A mutant. This change in the protonation extent leads to the qualitative change in the spectral characteristics of the protein (Fig. 2). Before the light irradiation, the protein absorbed in the near UV range with a maximum at 348 nm (Table) and was colorless because the protonated form of the protein barely fluoresces. It should be noted for comparison that the wild-type protein before photoconversion has typically green fluorescence with an absorption maximum at 494 nm and a maximum of fluorescence emission at 506 nm. After the light irradiation, the absorption maximum of DendFP-S142A shifted to 455 nm, and the fluores-
EXPERIMENTAL Gene Expression and Isolation of Recombinant Proteins E. coli JM-109 (DE3) cells were transformed with the pQE-30 plasmid containing the target protein gene, which was kindly provided by K.A. Lukyanov
Spectral properties of DendFP-S142A before and after photoactivation Absorption maxima, nm
Before photoactivation After photoactivation
Emission maxima, nm
phenol (protonated) form
phenolate (anionic) form
phenol (protonated) form
phenolate (anionic) form
384
506*
455
512
455; 485
575*
501; 536
* Minor peak. ** On the shoulder of the main peak. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 3
2017
576**
342
PAKHOMOV et al.
(а)
(b) 455; 485 nm 501; 536 nm
1.0
1.6 1.4
0.8
1.0
Fluorescence
Absorption
1.2
384 nm
0.8
455 nm
0.6 0.4
0.6 0.4 0.2
0.2 0
0 300
400 500 Wavelength, nm
600
300
400 500 Wavelength, nm
(c)
600
700
(d) 1.0
1.0
0.8
0.6
0.6
Fluorescence
Fluorescence
1 0.8
0.4 2
0.2 0
2
0.4
1
0.2 0
0
100 Time, s
200
0
100 Time, s
200
Fig. 2. Spectral properties of DendFP-S142A. The absorption spectrum before (black curve) and after (gray curve) photoconversion (a). The fluorescent excitation (black curve) and emission (gray curve) spectra of photoactivatable protein (b). The growth of green fluorescence (curve 1) is accompanied by the insignificant growth of red fluorescence (curve 2) when DendFP-S142A is irradiated by blue UV light (405 nm) (c). For comparison, the irradiation of wild-type DendFP leads to a decrease of green (curve 1) and growth of red (curve 2) fluorescence (d).
(Institute of Bioorganic Chemistry, RAS). The transformed cells were grown in the LB nutrient medium containing ampicillin (100 μg/mL) at 37°C under stirring (200 rpm) overnight. For a complete protein maturation, cells were further incubated at room temperature and stirred for another two days. The recombinant proteins containing the 6x(His) sequence at the N-termini were isolated from the cellular lysate by metal-affinity chromatography on a Ni-NTA agarose (Qiagen, United States) according to the manufacturer’s protocol. Site-Directed Mutagenesis was performed using a QuikChange kit (Stratagene, United States). Absorption Spectra were recorded on a Cary 50 Bio spectrophotometer (Varian, Inc., United States); Fluorescence Spectra were recorded on a Cary Eclipse spectrofluorometer (Varian, Inc., United States).
Photoconversion of the protein was carried out at 4°C using a CL-215 UV transilluminator (365 nm, Ultra-Violet Products, Inc., United States). To monitor the increase and decrease of fluorescence during photoconversion, the protein was immobilized on NiNTA agarose. The sorbent beads were visualized on a Leica DMI6000 inverted fluorescence microscope. Photoconversion was induced by the irradiation of the protein with violet light through a 63× objective using a cube with color filters for blue FP. The dynamics of green and red fluorescence was observed using a cube with color filters for GFP and TexasRed, respectively. ACKNOWLEDGMENTS The work was supported by the Russian Science Foundation (project no. 14-50-00131).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 3
2017
GENERATION OF PHOTOACTIVATABLE FLUORESCENT PROTEIN
REFERENCES 1. Chudakov, D.M., Matz, M.V., Lukyanov, S., and Lukyanov, K.A., Physiol. Rev., 2010, vol. 90, pp. 1103– 1163. 2. Day, R.N. and Davidson, M.W., Chem. Soc. Rev., 2009, vol. 38, pp. 2887–2921. 3. Stepanenko, O.V., Stepanenko, O.V., Shcherbakova, D.M., Kuznetsova, I.M., Turoverov, K.K., and Verkhusha, V.V., BioTechniques, 2011, vol. 51, pp. 313–327. 4. Pakhomov, A.A. and Martynov, V.I., Chem. Biol., 2008, vol. 15, pp. 755–764. 5. Lukyanov, K.A., Chudakov, D.M., Lukyanov, S., and Verkhusha, V.V., Nat. Rev. Mol. Cell Biol., 2005, vol. 6, pp. 885–891. 6. Shcherbakova, D.M., Sengupta, P., LippincottSchwartz, J., and Verkhusha, V.V., Annu. Rev. Biophys., 2014, vol. 43, pp. 303–329. 7. Patterson, G.H. and Lippincott-Schwartz, J., Science, 2002, vol. 297, pp. 1873–1877. 8. Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., and Lukyanov, K.A., Nat. Biotechnol., 2003, vol. 21, pp. 191–194. 9. Habuchi, S., Ando, R., Dedecker, P., Verheijen, W., Mizuno, H., Miyawaki, A., and Hofkens, J., Proc. Natl. Acad. Sci. U. S. A., 2005, vol. 102, pp. 9511–9516. 10. Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., and Miyawaki, A., Proc. Natl. Acad. Sci. U. S. A., 2002, vol. 99, pp. 12651–12656. 11. Mizuno, H., Mal, T.K., Tong, K.I., Ando, R., Furuta, T., Ikura, M., and Miyawaki, A., Mol. Cell, 2003, vol. 12, pp. 1051–1058. 12. Labas, Y.A., Gurskaya, N.G., Yanushevich, Y.G., Fradkov, A.F., Lukyanov, K.A., Lukyanov, S.A., and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
13. 14.
15.
16. 17. 18. 19. 20.
21. 22.
343
Matz, M.V., Proc. Natl. Acad. Sci. U. S. A., 2002, vol. 99, pp. 4256–4261. Pakhomov, A.A., Martynova, N.Y., Gurskaya, N.G., Balashova, T.A., and Martynov, V.I., Biochemistry (Moscow), 2004, vol. 69, pp. 901–908. Pletneva, N.V., Pletnev, S., Pakhomov, A.A., Chertkova, R.V., Martynov, V.I., Muslinkina, L., Dauter, Z., and Pletnev, V.Z., Acta Crystallogr. D Struct. Biol., 2016, vol. 72, pp. 922–932. Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.D., and Nienhaus, G.U., Proc. Natl. Acad. Sci. U. S. A., 2004, vol. 101, pp. 15905–15910. Nienhaus, K., Nienhaus, G.U., Wiedenmann, J., and Nar, H., Proc. Natl. Acad. Sci. U. S. A., 2005, vol. 102, pp. 9156–9159. Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., and Miyawaki, A., EMBO Rep., 2005, vol. 6, pp. 233– 238. Habuchi, S., Tsutsui, H., Kochaniak, A.B., Miyawaki, A., and van Oijen, A.M., PLoS One, 2008, vol. 3. Hoi, H., Shaner, N.C., Davidson, M.W., Cairo, C.W., Wang, J., and Campbell, R.E., J. Mol. Biol., 2010, vol. 401, pp. 776–791. McEvoy, A.L., Hoi, H., Bates, M., Platonova, E., Cranfill, P.J., Baird, M.A., Davidson, M.W., Ewers, H., Liphardt, J., and Campbell, R.E., PLos One, 2012, vol. 7. Pakhomov, A.A., Chertkova, R.V., and Martynov, V.I., Russ. J. Bioorg. Chem., 2015, vol. 41, pp. 602–606. Meech, S.R., Chem. Soc. Rev., 2009, vol. 38, pp. 2922– 2934.
Translated by A. Levina
Vol. 43
No. 3
2017
