Лукьянов Константин Анатольевич


Период обученияСтрана, городУчебное заведениеДополнительная информация
1986–1991 Москва, Россия Биологический факультет МГУ

Премии и заслуги

1. Премии Международной академической издательской компании “Наука” за лучшую публикацию в издаваемых ею журналах за 1996, 1999 и 2011 гг.

2. Медаль Российской академии наук с премией для молодых ученых РАН, 2000 г.

3. Scopus Award Russia 2013 за выдающийся вклад в развитие науки в области биологии на национальном и международном уровнях.

4. Награда “Highly cited researcher – Russia 2015” от Thomson Reuters по данным Web of Science Core Collection и Essential Science Indicators.

Избранные публикации

  1. Pennacchietti F., Serebrovskaya E.O., Faro A.R., Shemyakina I.I., Bozhanova N.G., Kotlobay A.A., Gurskaya N.G., Bodén A., Dreier J., Chudakov D.M., Lukyanov K.A., Verkhusha V.V., Mishin A.S., Testa I. (2018). Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy. Nat. Methods 15 (8), 601–604 [+]

    Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.

  2. Shcheslavskiy V.I., Shirmanova M.V., Dudenkova V.V., Lukyanov K.A., Gavrina A.I., Shumilova A.V., Zagaynova E., Becker W. (2018). Fluorescence time-resolved macroimaging. Opt Lett 43 (13), 3152–3155 [+]

    While laser scanning fluorescence lifetime imaging (FLIM) is a powerful approach for cell biology, its small field of view (typically less than 1 mm) makes it impractical for the imaging of large biological samples that is often required for biomedical applications. Here we present a system that allows performing FLIM on macroscopic samples as large as 18 mm with a lateral resolution of 15 μm. The performance of the system is verified with FLIM of endogenous metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and genetically encoded fluorescent protein mKate2 in a mouse tumor in vivo.

  3. Lidsky P.V., Lukyanov K.A., Misra T., Handke B., Mishin A.S., Lehner C.F. (2018). A genetically encoded fluorescent probe for imaging of oxygenation gradients in living Drosophila. Development 145 (4), dev156257 [+]

    Oxygen concentrations vary between tissues of multicellular organisms and change under certain physiological or pathological conditions. Multiple methods have been developed for measuring oxygenation of biological samples and However, most require complex equipment, are laborious and have significant limitations. Here we report that oxygen concentration determines the choice between two maturation pathways of DsRed FT (Timer). At high oxygen levels, this DsRed derivate matures predominantly into a red fluorescent isoform. By contrast, a green fluorescent isoform is favored by low oxygen levels. Ratiometric analysis of green and red fluorescence after a pulse of Timer expression in larvae provides a record of the history of tissue oxygenation during a subsequent chase period, for the whole animal with single-cell precision. Tissue spreads revealed fine differences in oxygen exposure among different cells of the same organ. We expect that the simplicity and robustness of our approach will greatly impact hypoxia research, especially in small animal models.

  4. Povarova N.V., Markina N.M., Baranov M.S., Barinov N.A., Klinov D.V., Kozhemyako V.B., Lukyanov K.A. (2018). A water-soluble precursor for efficient silica polymerization by silicateins. Biochem. Biophys. Res. Commun. 495 (2), 2066–2070 [+]

    Silicateins, the spicule-forming proteins from marine demosponges capable to polymerize silica, are popular objects of biomineralization studies due to their ability to form particles varied in shape and composition under physiological conditions. Despite the occurrence of the many approaches to nanomaterial synthesis using silicateins, biochemical properties of this protein family are poorly characterized. The main reason for this is that tetraethyl orthosilicate (TEOS), the commonly used silica acid precursor, is almost insoluble in water and thus is poorly available for the protein. To solve this problem, we synthesized new water-soluble silica precursor, tetra(glycerol)orthosilicate (TGS), and characterized biochemical properties of the silicatein A1 from marine sponge Latrunculia oparinae. Compared to TEOS, TGS ensured much greater activity of silicatein and was less toxic for the mammalian cell culture. We evaluated optimum conditions for the enzyme - pH range, temperature and TGS concentration. We concluded that TGS is a useful silica acid precursor that can be used for silica particles synthesis and in vivo applications.

  5. Markina N.M., Pereverzev A.P., Staroverov D.B., Lukyanov K.A., Gurskaya N.G. (2018). Generation of Cell Lines Stably Expressing a Fluorescent Reporter of Nonsense-Mediated mRNA Decay Activity. Methods Mol. Biol. 1720, 187–204 [+]

    Nonsense-mediated mRNA decay (NMD) is a mechanism of mRNA surveillance ubiquitous among eukaryotes. Importantly, NMD not only removes aberrant transcripts with premature stop codons, but also regulates expression of many normal genes. A recently introduced dual-color fluorescent protein-based reporter enables analysis of NMD activity in live cells. In this chapter we describe the method to generate stable transgenic cell lines expressing the splicing-dependent NMD reporter using consecutive steps of lentivirus transduction and Tol2 transposition.

  6. Pakhomov A.A., Martynov V.I., Orsa A.N., Bondarenko A.A., Chertkova R.V., Lukyanov K.A., Petrenko A.G., Deyev I.E. (2017). Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor. Biochem. Biophys. Res. Commun. 493 (4), 1518–1521 [+]

    Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

  7. Bozhanova N.G., Baranov M.S., Klementieva N.V., Sarkisyan K.S., Gavrikov A.S., Yampolsky I.V., Zagaynova E.V., Lukyanov S.A., Lukyanov K.A., Mishin A.S. (2017). Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal. Chemical Science 8 (10), 7138–7142 [+]

    We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.

  8. Povarova N.V., Petri N.D., Blokhina A.E., Bogdanov A.M., Gurskaya N.G., Lukyanov K.A. (2017). Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models. Int J Mol Sci 18 (7), [+]

    Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed "trio-FPs" consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

  9. Bozhanova N.G., Baranov M.S., Sarkisyan K.S., Gritcenko R., Mineev K.S., Golodukhina S.V., Baleeva N.S., Lukyanov K.A., Mishin A.S. (2017). Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores. ACS Chem. Biol. 12 (7), 1867–1873 [+]

    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.

  10. Sergeeva T.F., Shirmanova M.V., Zlobovskaya O.A., Gavrina A.I., Dudenkova V.V., Lukina M.M., Lukyanov K.A., Zagaynova E.V. (2017). Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis. Biochim. Biophys. Acta 1864 (3), 604–611 [+]

    A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.

  11. Acharya A., Bogdanov A.M., Grigorenko B.L., Bravaya K.B., Nemukhin A.V., Lukyanov K.A., Krylov A.I. (2017). Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem. Rev. 117 (2), 758–795 [+]

    Photoinduced reactions play an important role in the photocycle of fluorescent proteins from the green fluorescent protein (GFP) family. Among such processes are photoisomerization, photooxidation/photoreduction, breaking and making of covalent bonds, and excited-state proton transfer (ESPT). Many of these transformations are initiated by electron transfer (ET). The quantum yields of these processes vary significantly, from nearly 1 for ESPT to 10(-4)-10(-6) for ET. Importantly, even when quantum yields are relatively small, at the conditions of repeated illumination the overall effect is significant. Depending on the task at hand, fluorescent protein photochemistry is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. The phenomena arising due to phototransformations include (i) large Stokes shifts, (ii) photoconversions, photoactivation, and photoswitching, (iii) phototoxicity, (iv) blinking, (v) permanent bleaching, and (vi) formation of long-lived intermediates. The focus of this review is on the most recent experimental and theoretical work on photoinduced transformations in fluorescent proteins. We also provide an overview of the photophysics of fluorescent proteins, highlighting the interplay between photochemistry and other channels (fluorescence, radiationless relaxation, and intersystem crossing). The similarities and differences with photochemical processes in other biological systems and in dyes are also discussed.

  12. Klementieva N.V., Pavlikov A.I., Moiseev A.A., Bozhanova N.G., Mishina N.M., Lukyanov S.A., Zagaynova E.V., Lukyanov K.A., Mishin A.S. (2017). Intrinsic blinking of red fluorescent proteins for super-resolution microscopy. Chem. Commun. (Camb.) 53 (5), 949–951 [+]

    Single-molecule localization microscopy relies on either controllable photoswitching of fluorescent probes or their robust blinking. We have found that blinking of monomeric red fluorescent proteins TagRFP, TagRFP-T, and FusionRed occurs at moderate illumination power and matches well with camera acquisition speed. It allows for super-resolution image reconstruction of densely labelled structures in live cells using various algorithms.

  13. Kost L.A., Nikitin E.S., Ivanova V.O., Sung U., Putintseva E.V., Chudakov D.M., Balaban P.M., Lukyanov K.A., Bogdanov A.M. (2017). Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor. PLoS ONE 12 (9), e0184225 [+]

    Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

  14. Мамонтова А.В., Григорьев А.П., Царькова А.С., Лукьянов К.А., Богданов А.М. (2017). БОРЬБА ЗА ФОТОСТАБИЛЬНОСТЬ: МЕХАНИЗМЫ ОБЕСЦВЕЧИВАНИЯ ФЛУОРЕСЦЕНТНЫХ БЕЛКОВ. Биоорг. хим. 43 (6), 598–607 [+]

    Современная биологическая наука нуждается в визуализации исследуемых объектов на уровнях клетки, органелл и отдельных молекул. Заметное место среди способов визуализации занимают методы, основанные на регистрации флуоресценции флуорофоров, которыми помечены объекты исследования. Флуоресцентные белки (ФБ) весьма популярны в качестве генетически кодируемых флуоресцентных меток для прижизенной визуализации целевых структур и процессов в живых системах. Одной из ключевых характеристик ФБ является их фотостабильность, т.е. устойчивость к фотохимическим реакциям, приводящим к исчезновению флуоресцентного сигнала. В данном обзоре описаны известные на данный момент молекулярные механизмы, лежащие в основе фотообесцвечивания, и методы, применяемые для улучшения фотостабильности флуоресцентных белков

  15. Klementieva N.V., Lukyanov K.A., Markina N.M., Lukyanov S.A., Zagaynova E.V., Mishin A.S. (2016). Green-to-red primed conversion of Dendra2 using blue and red lasers. Chem. Commun. (Camb.) 52 (89), 13144–13146 [+]

    Recently, an unusual phenomenon of primed conversion of fluorescent protein Dendra2 by combined action of blue (488 nm) and near-infrared (700-780 nm) lasers was discovered. Here we demonstrate that primed conversion can be induced by red lasers (630-650 nm) common for most confocal and single molecule detection microscopes.

  16. Klementieva N.V., Snopova L.B., Prodanets N.N., Furman O.E., Dudenkova V.V., Zagaynova E.V., Lukyanov K.A., Mishin A.S. (2016). Fluorescence Imaging of Actin Fine Structure in Tumor Tissues Using SiR-Actin Staining. Anticancer Res. 36 (10), 5287–5294 [+]


    The rearrangement of actin cytoskeleton is being increasingly considered a marker of cancer cell activity, but the fine structure and remodeling of microfilaments within tumor tissue still remains unclear.


    We used the recently introduced silicon-rhodamine (SiR)-actin dye to visualize endogenous actin within tissues by confocal or total internal reflection fluorescence microscopy. We established imaging conditions for robust blinking of SiR-actin, which makes this dye applicable for super-resolution localization microscopy, as well as for an efficient background elimination.


    We studied tumor tissue samples in two mouse models at high resolution and revealed a complex network of thick curved bundles of actin in cancer cells in tumors. This actin pattern differed strongly from that in cancer cells in vitro and in normal tissues.


    Localization microscopy with SiR-actin provides an efficient way to visualize fine actin structure in tumor tissues. It is potentially applicable to a variety of biological and clinical samples.

  17. Ryumina A.P., Serebrovskaya E.O., Staroverov D.B., Zlobovskaya O.A., Shcheglov A.S., Lukyanov S.A., Lukyanov K.A. (2016). Lysosome-associated miniSOG as a photosensitizer for mammalian cells. BioTechniques 61 (2), 92–4 [+]

    Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis.

  18. Sarkisyan K.S., Bolotin D.A., Meer M.V., Usmanova D.R., Mishin A.S., Sharonov G.V., Ivankov D.N., Bozhanova N.G., Baranov M.S., Soylemez O., Bogatyreva N.S., Vlasov P.K., Egorov E.S., Logacheva M.D., Kondrashov A.S., Chudakov D.M., Putintseva E.V., Mamedov I.Z., Tawfik D.S., Lukyanov K.A., Kondrashov F.A. (2016). Local fitness landscape of the green fluorescent protein. Nature 533 (7603), 397–401 [+]

    Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

  19. Bogdanov A.M., Acharya A., Titelmayer A.V., Mamontova A.V., Bravaya K.B., Kolomeisky A.B., Lukyanov K.A., Krylov A.I. (2016). Turning On and Off Photoinduced Electron Transfer in Fluorescent Proteins by π-Stacking, Halide Binding, and Tyr145 Mutations. J. Am. Chem. Soc. 138 (14), 4807–17 [+]

    Photoinduced electron transfer in fluorescent proteins from the GFP family can be regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. Photooxidation commonly results in green-to-red photoconversion called oxidative redding. We discovered that yellow FPs do not undergo redding; however, the redding is restored upon halide binding. Calculations of the energetics of one-electron oxidation and possible electron transfer (ET) pathways suggested that excited-state ET proceeds through a hopping mechanism via Tyr145. In YFPs, the π-stacking of the chromophore with Tyr203 reduces its electron-donating ability, which can be restored by halide binding. Point mutations confirmed that Tyr145 is a key residue controlling ET. Substitution of Tyr145 by less-efficient electron acceptors resulted in highly photostable mutants. This strategy (i.e., calculation and disruption of ET pathways by mutations) may represent a new approach toward enhancing photostability of FPs.

  20. Povarova N.V., Bozhanova N.G., Sarkisyan K.S., Gritcenko R., Baranov M.S., Yampolsky I.V., Lukyanov K.A., Mishin A.S. (2016). Docking-guided identification of protein hosts for GFP chromophore-like ligands. J. Mater. Chem. C 4, 3036–3040 [+]

    Synthetic analogs of the Green Fluorescent Protein (GFP) chromophore emerge as promising fluorogenic dyes for labeling in living systems. Here, we report the computational identification of protein hosts capable of binding to and enhancing fluorescence of GFP chromophore derivatives. Automated docking of GFP-like chromophores to over 3000 crystal structures of Escherichia coli proteins available in the Protein Data Bank allowed the identification of a set of candidate proteins. Four of these proteins were tested experimentally in vitro for binding with the GFP chromophore and its red-shifted Kaede chromophore-like analogs. Two proteins were found to possess sub-micromolar affinity for some Kaede-like chromophores and activate fluorescence of these fluorogens.

  21. Zlobovskaya O.A., Sergeeva T.F., Shirmanova M.V., Dudenkova V.V., Sharonov G.V., Zagaynova E.V., Lukyanov K.A. (2016). Genetically encoded far-red fluorescent sensors for caspase-3 activity. BioTechniques 60 (2), 62–8 [+]

    Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.

  22. Prudkovsky A.A., Ivanenko V.N., Nikitin M.A., Lukyanov K.A., Belousova A., Reimer J.D., Berumen M.L. (2016). Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea. PLoS ONE 11 (2), e0146861 [+]

    Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

  23. Eroshkin F.M., Nesterenko A.M., Borodulin A.V., Martynova N.Y., Ermakova G.V., Gyoeva F.K., Orlov E.E., Belogurov A.A. Jr, Lukyanov K.A., Bayramov A.V., Zaraisky A.G. (2016). Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis. Sci Rep 6, 23049 [+]

    Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

  24. Gurskaya N.G., Staroverov D.B., Lukyanov K.A. (2016). Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells. Meth. Enzymol. 572, 255–68 [+]

    Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines.

  25. Gurskaya N.G., Pereverzev A.P., Staroverov D.B., Markina N.M., Lukyanov K.A. (2016). Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins. Meth. Enzymol. 572, 291–314 [+]

    Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mechanism of specific degradation of transcripts with a premature stop codon. NMD eliminates aberrant mRNAs arising from mutations, alternative splicing, and other events in cells. In addition, many normal transcripts undergo NMD. Recent studies demonstrated that NMD activity is specifically regulated and that NMD can play a role of global regulator of gene expression. Recently, we developed dual-color fluorescent protein-based reporters for quantification of NMD activity using fluorescence microscopy and flow cytometry (Pereverzev, Gurskaya, et al., 2015). Due to ratiometric fluorescence response, these reporters make it possible to assess NMD activity in live cells at the single-cell level and to reveal otherwise hidden heterogeneity of cells in respect of NMD activity. Here we provide a detailed description of applications of the NMD reporters in mammalian cell lines.

  26. Pereverzev A.P., Matlashov M.E., Staroverov D.B., Lukyanov K.A., Gurskaya N.G. (2015). Differences of Nonsense-Mediated mRNA Degradation Activity in Mammalian Cell Lines Revealed by a Fluorescence Reporter. Bioorg. Khim. 41 (5), 587–91 [+]

    Activity of nonsense-mediated mRNA degradation (NMD) was studied in several mammalian cell cultures using recently developed genetically encoded fluorescence sensor [Pereverzev et al., Sci. Rep., 2015, vol. 5, p. 7729]. This NMD reporter enables measurement of NMD activity in single live cells using ratio of green and red fluorescent proteins signals. The following cell lines were analyzed: mouse colon carcinoma CT26, mouse Lewis lung carcinoma LLC, human T-cell leukemia Jurkat, and spontaneously immortalized human keratinocytes HaCaT. These cell lines demonstrated very different NMD activities. In CT26, NMD activity was low, whereas in LLC it was high (8.5-fold higher than in CT26). Jurkat and HaCaT cells possessed strong heterogeneity and consisted of two cell subpopulations with high and low NMD activities. In addition, we detected high NMD activity in primary culture of mouse embryonic hippocampal neurons.

  27. Pletnev V.Z., Pletneva N.V., Sarkisyan K.S., Mishin A.S., Lukyanov K.A., Goryacheva E.A., Ziganshin R.H., Dauter Z., Pletnev S. (2015). Structure of the green fluorescent protein NowGFP with an anionic tryptophan-based chromophore. Acta Crystallogr. D Biol. Crystallogr. 71 (Pt 8), 1699–707 [+]
  28. Walker C.L., Lukyanov K.A., Yampolsky I.V., Mishin A.S., Bommarius A.S., DurajThatte A.M., Azizi B., Tolbert L.M., Solntsev K.M. (2015). Fluorescence imaging using synthetic GFP chromophores. Curr Opin Chem Biol 27, 64–74 [+]

    Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.

  29. Yuzhakova D.V., Shirmanova M.V., Serebrovskaya E.O., Lukyanov K.A., Druzhkova I.N., Shakhov B.E., Lukyanov S.A., Zagaynova E.V. (2015). CT26 murine colon carcinoma expressing the red fluorescent protein KillerRed as a highly immunogenic tumor model. J Biomed Opt 20 (8), 88002 [+]

    The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.

  30. Sarkisyan K.S., Goryashchenko A.S., Lidsky P.V., Gorbachev D.A., Bozhanova N.G., Gorokhovatsky A.Y., Pereverzeva A.R., Ryumina A.P., Zherdeva V.V., Savitsky A.P., Solntsev K.M., Bommarius A.S., Sharonov G.V., Lindquist J.R., Drobizhev M., Hughes T.E., Rebane A., Lukyanov K.A., Mishin A.S. (2015). Green Fluorescent Protein with Anionic Tryptophan-Based Chromophore and Long Fluorescence Lifetime. Biophys. J. 109 (2), 380–9 [+]

    Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

  31. Mishin A.S., Belousov V.V., Solntsev K.M., Lukyanov K.A. (2015). Novel uses of fluorescent proteins. Curr Opin Chem Biol 27, 1–9 [+]The field of genetically encoded fluorescent probes is developing rapidly. New chromophore structures were characterized in proteins of green fluorescent protein (GFP) family. A number of red fluorescent sensors, for example, for pH, Ca(2+) and H2O2, were engineered for multiparameter imaging. Progress in development of microscopy hardware and software together with specially designed FPs pushed superresolution fluorescence microscopy towards fast live-cell imaging. Deeper understanding of FPs structure and photophysics led to further development of imaging techniques. In addition to commonly used GFP-like proteins, unrelated types of FPs on the base of flavin-binding domains, bilirubin-binding domains or biliverdin-binding domains were designed. Their distinct biochemical and photophysical properties opened previously unexplored niches of FP uses such as labeling under anaerobic conditions, deep tissue imaging and even patients' blood analysis. ID:1293
  32. Zlobovskaya O.A., Sarkisyan K.S., Lukyanov K.A. (2015). Infrared Fluorescent Protein iRFP as an Acceptor for Förster Resonance Energy Transfer. Bioorg. Khim. 41 (3), 299–304 [+]

    Bacteriophytochrome-based infrared fluorescent protein iRFP was tested as an acceptor for F6rster resonance energy transfer (FRET). Far-red GFP-like fluorescent proteins mKate2, eqFP650, and eqFP670 were used as donors; Bacterial expression vectors encoding donor and acceptor proteins fused by a 17-amino acid linker were.constructed. FRET for purified proteins in vitro was, estimated from increase of the donor emission after digestion of the linker. Among the three constructs tested, the most efficient FRET (approximately 30%) was detected for the eqFP650-iRFP pair.

  33. Povarova N.V., Baranov M.S., Kovalchuk S.N., Semiletova I.V., Lukyanov K.A., Kozhemyak V.B. (2015). Novel Water-Soluble Substrate for Silicateins. Bioorg. Khim. 41 (3), 380–2 [+]

    We suggested to use tetrakis(2-hydroxyethyl)orthosilicate (THEOS) as a substrate for silicateins--an enzyme family playing a key role in formation of skeleton in marine sponges. We compared THEOS with tetraethylorthosilicate (TEOS)--a commonly used substrate for silicateins. These substrates were tested in reaction of amorphous silica formation in vitro catalyzed by silicatein Al from sponge Latrunculia oparinae. It was found that reaction with THEOS occurs more efficiently than with TEOS, probably due to high water solubility and higher hydrolysis rate of THEOS.

  34. Mamontova A.V., Bogdanov A.M., Lukyanov K.A. (2015). Influence of cell growth conditions and medium composition on EGFP photostability in live cells. BioTechniques 58 (5), 258–261 [+]

    Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham's F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4, cyanocobalamine, lipoic acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media.

  35. Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G., Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5, 7729 [+]

    Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.

  36. Luker K.E., Pata P., Shemiakina I.I., Pereverzeva A., Stacer A.C., Shcherbo D.S., Pletnev V.Z., Skolnaja M., Lukyanov K.A., Luker G.D., Pata I., Chudakov D.M. (2015). Comparative study reveals better far-red fluorescent protein for whole body imaging. Sci Rep 5, 10332 [+]
  37. Sarkisyan K.S., Zlobovskaya O.A., Gorbachev D.A., Bozhanova N.G., Sharonov G.V., Staroverov D.B., Egorov E.S., Ryabova A.V., Solntsev K.M., Mishin A.S., Lukyanov K.A. (2015). KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS ONE 10 (12), e0145287 [+]

    Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.

  38. Pletneva N.V., Pletnev V.Z., Sarkisyan K.S., Gorbachev D.A., Egorov E.S., Mishin A.S., Lukyanov K.A., Dauter Z., Pletnev S. (2015). Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore. PLoS ONE 10 (12), e0145740 [+]

    Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.

  39. Shirmanova M., Yuzhakova D., Snopova L., Perelman G., Serebrovskaya E., Lukyanov K., Turchin I., Subochev P., Lukyanov S., Kamensky V., Zagaynova E. (2015). Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model. PLoS ONE 10 (12), e0144617 [+]

    The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

  40. Serebrovskaya E.O., Ryumina A.P., Boulina M.E., Shirmanova M.V., Zagaynova E.V., Bogdanova E.A., Lukyanov S.A., Lukyanov K.A. (2014). Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed. J Biomed Opt 19 (7), 071403 [+]

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  41. Ryumina A.P., Serebrovskaya E.O., Shirmanova M.V., Snopova L.B., Kuznetsova M.M., Turchin I.V., Ignatova N.I., Klementieva N.V., Fradkov A.F., Shakhov B.E., Zagaynova E.V., Lukyanov K.A., Lukyanov S.A. (2013). Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells. Biochim. Biophys. Acta 1830 (11), 5059–67 [+]

    Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells.

  42. Pletnev V.Z., Pletneva N.V., Lukyanov K.A., Souslova E.A., Fradkov A.F., Chudakov D.M., Chepurnykh T., Yampolsky I.V., Wlodawer A., Dauter Z., Pletnev S. (2013). Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 9), 1850–60 [+]
  43. Baranov M.S., Solntsev K.M., Lukyanov K.A., Yampolsky I.V. (2013). A synthetic approach to GFP chromophore analogs from 3-azidocinnamates. Role of methyl rotors in chromophore photophysics. Chem. Commun. (Camb.) 49 (51), 5778–80 [+]

    We have suggested a novel combinatorial approach for synthesis of otherwise inaccessible GFP chromophore analogs, and studied the influence of aliphatic substituents on their pH-dependent spectral properties. We found that the demethylation at C or N positions of the imidazolone ring leads to a decrease in the excited state lifetime.

  44. Lukyanov K.A., Belousov V.V. (2013). Genetically encoded fluorescent redox sensors. Biochim. Biophys. Acta , [+]

    Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes.

  45. Baranov M.S., Lukyanov K.A., Yampolsky I.V. (2013). Synthesis of the chromophores of fluorescent proteins and their analogs. Russ. J. Bioorgan. Chem. 39 (3), 223–244 [+]

    Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4H-imidazol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroaminoacids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroaminoacids, and in the third part we summarize cyclizations of N-acyldehydroaminoacids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.

  46. Baranov M.S., Lukyanov K.A., Ivashkin P.E., Yampolsky I.V. (2013). Efficient synthetic approach to fluorescent oxazole-4-carboxylate derivatives. Synt. Comm. 43 (17), 2337–2342 ID:1041
  47. Baranov M.S., Lukyanov K.A., Borissova A.O., Shamir J., Kosenkov D., Slipchenko L.V., Tolbert L.M., Yampolsky I.V., Solntsev K.M. (2012). Conformationally locked chromophores as models of excited-state proton transfer in fluorescent proteins. J. Am. Chem. Soc. 134 (13), 6025–32 [+]

    Members of the green fluorescent protein (GFP) family form chromophores by modifications of three internal amino acid residues. Previously, many key characteristics of chromophores were studied using model compounds. However, no studies of intermolecular excited-state proton transfer (ESPT) with GFP-like synthetic chromophores have been performed because they either are nonfluorescent or lack an ionizable OH group. In this paper we report the synthesis and photochemical study of two highly fluorescent GFP chromophore analogues: p-HOBDI-BF2 and p-HOPyDI:Zn. Among known fluorescent compounds, p-HOBDI-BF(2) is the closest analogue of the native GFP chromophore. These irrreversibly (p-HOBDI-BF(2)) and reversibly (p-HOPyDI:Zn) locked compounds are the first examples of fully planar GFP chromophores, in which photoisomerization-induced deactivation is suppressed and protolytic photodissociation is observed. The photophysical behavior of p-HOBDI-BF2 and p-HOPyDI:Zn (excited state pK(a)'s, solvatochromism, kinetics, and thermodynamics of proton transfer) reveals their high photoacidity, which makes them good models of intermolecular ESPT in fluorescent proteins. Moreover, p-HOPyDI:Zn is a first example of "super" photoacidity in metal-organic complexes.

  48. Gurskaya N.G., Staroverov D.B., Zhang L., Fradkov A.F., Markina N.M., Pereverzev A.P., Lukyanov K.A. (2012). Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins. Nucleic Acids Res. 40 (8), e57 [+]

    Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

  49. Sarkisyan K.S., Yampolsky I.V., Solntsev K.M., Lukyanov S.A., Lukyanov K.A., Mishin A.S. (2012). Tryptophan-based chromophore in fluorescent proteins can be anionic. Sci Rep 2, 608 [+]

    Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with an anionic chromophore and key substitution Val61Lys was obtained. This is the first evidence strongly suggesting that tryptophan-based chromophores in fluorescent proteins can exist in an anionic charged state. Switching between protonated and deprotonated Trp66 in fluorescent proteins represents a new unexplored way to control their spectral properties.

  50. Ivashkin P.E., Lukyanov K.A., Yampolsky I.V. (2011). Synthesis of biosynthetic precursors of chromophores of red fluorescent proteins. Russ. J. Bioorgan. Chem. 37 (4), 411–420 [+]
  51. Ivashkin P.E., Lukyanov K.A., Lukyanov S., Yampolsky I.V. (2011). A synthetic GFP-like chromophore undergoes base-catalyzed autoxidation into acylimine red form. J. Org. Chem. 76 (8), 2782–91 [+]

    Fluorescent proteins are widely used in modern experimental biology, but much controversy exists regarding details of maturation of different types of their chromophores. Here we studied possible mechanisms of DsRed-type red chromophore formation using synthetic biomimetic GFP-like chromophores, bearing an acylamino substituent, corresponding to an amino acid residue at position 65. We have shown these model compounds to readily react with molecular oxygen to produce a highly unstable DsRed-like acylimine, isolated in the form of stable derivatives. Under the same aerobic conditions an unusual red-shifted imide chromophore--a product of 4-electron oxidation of Gly65 residue--is formed. Our data showed that GFP chromophore is prone to autoxidation at position 65 Cα by its chemical nature with basic conditions being the only key factor required.

  52. Serebrovskaya E.O., Gorodnicheva T.V., Ermakova G.V., Solovieva E.A., Sharonov G.V., Zagaynova E.V., Chudakov D.M., Lukyanov S., Zaraisky A.G., Lukyanov K.A. (2011). Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein. Biochem. J. 435 (1), 65–71 [+]

    Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.

  53. Shcherbo D., Shemiakina I.I., Ryabova A.V., Luker K.E., Schmidt B.T., Souslova E.A., Gorodnicheva T.V., Strukova L., Shidlovskiy K.M., Britanova O.V., Zaraisky A.G., Lukyanov K.A., Loschenov V.B., Luker G.D., Chudakov D.M. (2010). Near-infrared fluorescent proteins. Nat. Methods 7 (10), 827–9 [+]

    Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

  54. Lukyanov K.A., Serebrovskaya E.O., Lukyanov S., Chudakov D.M. (2010). Fluorescent proteins as light-inducible photochemical partners. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology , [+]

    Green Fluorescent Protein (GFP) and other related fluorescent proteins are generally used as genetically encoded, chemically inert labels in vivo. This review focuses on the emerging application of fluorescent proteins as light-inducible intracellular photochemical partners. The first example of a chemically active GFP-like protein was the phototoxic red fluorescent protein KillerRed, which can be used for precise light-induced killing of cells, protein inactivation, and studying reactive oxygen species signaling in different cellular compartments. Moreover, recent studies revealed that various GFPs can act as light-induced electron donors in photochemical reactions with biologically relevant electron acceptors. These findings have important implications for practical uses of fluorescent proteins as well as for our understanding of the evolution and biology of this protein family.

  55. Chudakov D.M., Matz M.V., Lukyanov S., Lukyanov K.A. (2010). Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90 (3), 1103–63 [+]

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

  56. Bogdanov A.M., Bogdanova E.A., Chudakov D.M., Gorodnicheva T.V., Lukyanov S., Lukyanov K.A. (2009). Cell culture medium affects GFP photostability: a solution. Nat. Methods 6 (12), 859–60 ID:298
  57. Pletnev S., Gurskaya N.G., Pletneva N.V., Lukyanov K.A., Chudakov D.M., Martynov V.I., Popov V.O., Kovalchuk M.V., Wlodawer A., Dauter Z., Pletnev V. (2009). Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J. Biol. Chem. 284 (46), 32028–39 [+]

    KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

  58. Yampolsky I.V., Balashova T.A., Lukyanov K.A. (2009). Synthesis and spectral and chemical properties of the yellow fluorescent protein zFP538 chromophore. Biochemistry 48 (33), 8077–82 [+]

    Members of the green fluorescent protein (GFP) family become chromophoric through a unique pathway based on autocatalytic modifications of their amino acid residues. The yellow fluorescent protein zFP538 from the button polyp Zoanthus possesses unique spectral characteristics that are intermediate between those of the green and orange-red fluorescent proteins. In this study, we used chemical synthesis to resolve conflicting data from crystallographic and biochemical analyses of the zFP538 chromophore structure. We synthesized 2-(5-amino-1-oxopentyl)-5-(4-hydroxybenzylidene)-3-methyl-3,5-dihydro-4H-imidazol-4-one (5), which can spontaneously react intramolecularly to form cyclic imine (7). Compound 7 represents the native chromophore structure reported in the crystallographic study. We have also discovered an unusual isomerization of a 2-acylimidazolone to a 2,6-diketopiperazine derivative. The zFP538 chromophore is a complex system with intriguing chemical and spectral behavior, properties that have led to discrepancies in the interpretation of its structure. Our study supports the findings of previous crystallographic work, which postulated a cyclic imine chromophore structure within the native zFP538 protein, and also provides an explanation for experimental results obtained in the biochemical characterization of zFP538-derived chromopeptides.

  59. Serebrovskaya E.O., Edelweiss E.F., Stremovskiy O.A., Lukyanov K.A., Chudakov D.M., Deyev S.M. (2009). Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein. Proc. Natl. Acad. Sci. U.S.A. 106 (23), 9221–5 [+]

    Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185(HER-2-ECD) antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185(HER-2-ECD)-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.

  60. Bogdanov A.M., Mishin A.S., Yampolsky I.V., Belousov V.V., Chudakov D.M., Subach F.V., Verkhusha V.V., Lukyanov S., Lukyanov K.A. (2009). Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol.  (5), 459–461 [+]

    Proteins of the green fluorescent protein (GFP) family are well known owing to their unique biochemistry and extensive use as in vivo markers. We discovered that GFPs of diverse origins can act as light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, via green-to-red GFP photoconversion, this process can be observed in living cells without additional treatment.

  61. Ivashkin P.E., Yampolsky I.V., Lukyanov K.A. (2009). Synthesis and properties of chromophores of fluorescent proteins. Russ. J. Bioorgan. Chem. 35 (6), 652–669 [+]

    We describe the existing approaches to the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones - model chromophores of fluorescent proteins and their nonnatural analogs. We discuss in detail the chemical (acid-base and redox reactions, cis-trans isomery, etc.) and spectral properties of the chromophores and the influence of substitutes and the environment. The study of synthetic chromophores allows for modeling of the photophysical characteristics of fluorescent proteins.

  62. Mishin A.S., Subach F.V., Yampolsky I.V., King W., Lukyanov K.A., Verkhusha V.V. (2008). The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Biochemistry 47 (16), 4666–73 [+]

    Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags.

  63. Yampolsky I.V., Kislukhin A.A., Amatov T.T., Shcherbo D., Potapov V.K., Lukyanov S., Lukyanov K.A. (2008). Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs. Bioorg. Chem. 36 (2), 96–104 [+]

    Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

  64. Shcherbo D., Merzlyak E.M., Chepurnykh T.V., Fradkov A.F., Ermakova G.V., Solovieva E.A., Lukyanov K.A., Bogdanova E.A., Zaraisky A.G., Lukyanov S., Chudakov D.M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4 (9), 741–6 [+]

    Разработан новый флуоресцентный белок Katushka, обладающий флуоресценцией в дальне-красной области спектра, которая является предпочтительной для анализа сигнала внутри тканей животных. Katushka в десять раз ярче, чем созданные ранее дальне-красные флуоресцентные белки и характеризуется высокой скоростью созревания, высокой рН-стабильностью и фотостабильностью. Это делает новый белок идеальным инструментом для прижизненного мечения клеток внутри целых организмов. Создан мономерный вариант белка Katushka, названный mKate, для исследования внутриклеточной локализации белков.

  65. Merzlyak E.M., Goedhart J., Shcherbo D., Bulina M.E., Shcheglov A.S., Fradkov A.F., Gaintzeva A., Lukyanov K.A., Lukyanov S., Gadella T.W., Chudakov D.M. (2007). Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4 (7), 555–7 [+]

    Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.

  66. Chudakov D.M., Lukyanov S., Lukyanov K.A. (2007). Using photoactivatable fluorescent protein Dendra2 to track protein movement. BioTechniques 42 (5), 553, 555, 557 passim [+]

    Photoactivatable fluorescent proteins are capable of dramatic changes in fluorescent properties in response to specific light irradiation. For example, they can be converted from cyan to green, or from green to red, or from nonfluorescent to a brightly fluorescent state. Several types of such proteins were developed recently, and some of them are already becoming popular tools to study protein mobility. Here we provide detailed recommendations on application of the monomeric green-to-red photoconvertible fluorescent protein Dendra2 for protein tracking in living cultured cells.

  67. Chudakov D.M., Lukyanov S., Lukyanov K.A. (2007). Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc 2 (8), 2024–32 [+]

    A number of photoactivatable GFP-like fluorescent proteins (PAFPs) have been reported whose fluorescence can be switched on or whose fluorescent state can be modified by relatively intense irradiation at a specific wavelength. The use of these proteins gives unique opportunities to photolabel and track fusion proteins in a living cell. Here, we provide a protocol for the primary visualization, photoactivation and tracking of two monomeric PAFPs recently developed in our lab. Both these proteins, PS-CFP2 and Dendra2, are fluorescent and can be visualized before photoactivation. Upon photoactivation, their excitation and emission spectra undergo a dramatic red shift. The brightness of their initial and photoconverted states, along with the high dynamic ranges of both proteins, make them an attractive tool for protein photolabeling. Excluding genetic constructs cloning, cell culturing and transfection, the whole protocol may take anywhere from 10 min to several hours, depending on motility of the protein being studied.

  68. Evdokimov A.G., Pokross M.E., Egorov N.S., Zaraisky A.G., Yampolsky I.V., Merzlyak E.M., Shkoporov A.N., Sander I., Lukyanov K.A., Chudakov D.M. (2006). Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7 (10), 1006–12 [+]

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

  69. Chudakov D.M., Chepurnykh T.V., Belousov V.V., Lukyanov S., Lukyanov K.A. (2006). Fast and precise protein tracking using repeated reversible photoactivation. Traffic 7 (10), 1304–10 [+]

    Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we report 'protein rivers tracking' technique based on repeated identical rounds of photoactivation and subsequent images averaging, which results in dramatic increase of imaging resolution for fast protein movement events.

  70. Belousov V.V., Fradkov A.F., Lukyanov K.A., Staroverov D.B., Shakhbazov K.S., Terskikh A.V., Lukyanov S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3 (4), 281–6 [+]

    Разработан уникальный флуоресцентный сенсор HyPer для прижизненного мониторинга колебаний концентрации одного из важнейших регуляторов биологических процессов — перекиси водорода. Имеющий белковую природу, HyPer может быть экспрессирован в клетках или направлен в определенный клеточный компартмент. Благодаря высокой специфичности и чувствительности, HyPer может быть использован для отслеживания колебаний концентрации перекиси водорода на уровне единственной клетки или клеточной органеллы.

  71. Gurskaya N.G., Verkhusha V.V., Shcheglov A.S., Staroverov D.B., Chepurnykh T.V., Fradkov A.F., Lukyanov S., Lukyanov K.A. (2006). Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24 (4), 461–5 [+]

    Разработан новый мономерный флуоресцентный белок Dendra, способный к необратимой фотоконверсии из зеленой флуоресцентной формы в красную. Белок Dendra обладает высокой яркостью флуоресценции и может быть активирован как ультрафиолетовым, так и синим светом.

  72. Bulina M.E., Lukyanov K.A., Britanova O.V., Onichtchouk D., Lukyanov S., Chudakov D.M. (2006). Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed. Nat Protoc 1 (2), 947–53 [+]

    The phototoxic red fluorescent GFP-like protein KillerRed has recently been described. The phototoxicity of KillerRed exceeds that of EGFP by at least 1,000-fold, making it the first fully genetically encoded photosensitizer. KillerRed opens up new possibilities for precise light-induced cell killing and target protein inactivation. Because KillerRed is encoded by a gene, it can be expressed in a spatially and temporally regulated manner, under a chosen promoter, and fused with the desired protein of interest or localization signal. Here we provide a protocol for target protein inactivation in cell culture using KillerRed. As KillerRed is a new tool, the protocol focuses on aspects that will allow users to maximize the potential of this protein, guiding the design of chimeric constructs, recommended control experiments and preferred illumination parameters. The protocol, which describes target protein visualization and subsequent inactivation, is a 2- or 3-d procedure.

  73. Bulina M.E., Chudakov D.M., Britanova O.V., Yanushevich Y.G., Staroverov D.B., Chepurnykh T.V., Merzlyak E.M., Shkrob M.A., Lukyanov S., Lukyanov K.A. (2006). A genetically encoded photosensitizer. Nat. Biotechnol. 24 (1), 95–9 [+]

    Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to beta-galactosidase and phospholipase Cdelta1 pleckstrin homology domain.

  74. Lukyanov K.A., Chudakov D.M., Fradkov A.F., Labas Y.A., Matz M.V., Lukyanov S. (2006). Discovery and properties of GFP-like proteins from nonbioluminescent anthozoa. Methods Biochem Anal 47, 121–38 ID:284
  75. Shkrob M.A., Yanushevich Y.G., Chudakov D.M., Gurskaya N.G., Labas Y.A., Poponov S.Y., Mudrik N.N., Lukyanov S., Lukyanov K.A. (2005). Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J. 392 (Pt 3), 649–54 [+]

    Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

  76. Chudakov D.M., Lukyanov S., Lukyanov K.A. (2005). Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23 (12), 605–13 [+]

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.

  77. Lukyanov K.A., Chudakov D.M., Lukyanov S., Verkhusha V.V. (2005). Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6 (11), 885–91 [+]

    The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.

  78. Yampolsky I.V., Remington S.J., Martynov V.I., Potapov V.K., Lukyanov S., Lukyanov K.A. (2005). Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata. Biochemistry 44 (15), 5788–93 [+]

    A model compound for the chromophore within the purple nonfluorescent GFP-like chromoprotein asFP595 was synthesized. The postulated structure of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone, was taken from the high-resolution crystal structure analysis of intact asFP595 [Quillin, M. L., Anstrom, D., Shu, X., O'Leary, S., Kallio, K., Lukyanov, K. A., and Remington, S. J. (2005) Kindling Fluorescent Protein from Anemonia sulcata: Dark-State Structure at 1.38 A Resolution, Biochemistry 44, 5774-5787]. Erlenmeyer lactonization and oxidation of the methylene group attached to the heteroaromatic moiety with selenium dioxide were used at the key stages of the synthesis. The spectral properties of the model chromophore in solution and their dependence on the pH and polarity of the solvent were investigated. In water, the chromophore was found to exist in two forms, neutral and anionic, with a pK(a) of 7.1. In a dimethylformamide solution, the spectral properties of the anionic form closely match those of the native protein, demonstrating that under these conditions, the compound is an excellent model for the chromophore within native asFP595.

  79. Chudakov D.M., Verkhusha V.V., Staroverov D.B., Souslova E.A., Lukyanov S., Lukyanov K.A. (2004). Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22 (11), 1435–9 [+]

    In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.

  80. Bulina M.E., Lukyanov K.A., Yampolsky I.V., Chudakov D.M., Staroverov D.B., Shcheglov A.S., Gurskaya N.G., Lukyanov S. (2004). New class of blue animal pigments based on Frizzled and Kringle protein domains. J. Biol. Chem. 279 (42), 43367–70 [+]

    The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds.

  81. Verkhusha V.V., Chudakov D.M., Gurskaya N.G., Lukyanov S., Lukyanov K.A. (2004). Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins. Chem. Biol. 11 (6), 845–54 [+]

    The mechanism of the chromophore maturation in members of the green fluorescent protein (GFP) family such as DsRed and other red fluorescent and chromoproteins was analyzed. The analysis indicates that the red chromophore results from a chemical transformation of the protonated form of the GFP-like chromophore, not from the anionic form, which appears to be a dead-end product. The data suggest a rational strategy to achieve the complete red chromophore maturation utilizing substitutions to favor the formation of the neutral phenol in GFP-like chromophore. Our approach to detect the neutral chromophore form expands the application of fluorescent timer proteins to faster promoter activities and more spectrally distinguishable fluorescent colors. Light sensitivity found in the DsRed neutral form, resulting in its instant transformation to the mature red chromophore, could be exploited to accelerate the fluorescence acquisition.

  82. Chudakov D.M., Lukyanov K.A. (2003). Use of green fluorescent protein (GFP) and its homologs for in vivo protein motility studies. Biochemistry Mosc. 68 (9), 952–7 [+]

    Green fluorescent protein (GFP) and its homologs are widely used as fluorescent markers of gene expression and for determination of protein localization and motility in living cells. In particular, based on GFP and GFP-like proteins a number of techniques have been developed that can be used either to estimate protein mobility in living cells, or to introduce a distinctive fluorescent signal in order to track the movement of labeled molecules directly. Considerable progress in the development of such technologies in the last two or three years motivates us to reevaluate the present scope of biotechnological instruments in studies of protein movement in cells.

  83. Gurskaya N.G., Fradkov A.F., Pounkova N.I., Staroverov D.B., Bulina M.E., Yanushevich Y.G., Labas Y.A., Lukyanov S., Lukyanov K.A. (2003). A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem. J. 373 (Pt 2), 403–8 [+]

    We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

  84. Bulina M.E., Verkhusha V.V., Staroverov D.B., Chudakov D.M., Lukyanov K.A. (2003). Hetero-oligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins. Biochem. J. 371 (Pt 1), 109–14 [+]

    The tendency for tetramerization is the main disadvantage in the green fluorescent protein homologues from Anthozoa species. We report a universal method called hetero-oligomeric tagging, which diminishes troublesome consequences of tetramerization of Anthozoa-derived fluorescent proteins (FP) in intracellular protein labelling. This approach is based on the co-expression of the FP-tagged protein of interest together with an excess of free non-fluorescent FP mutant. The resulting FP heterotetramers contain only a single target polypeptide and, therefore, can be considered pseudo-monomeric. Feasibility of the method has been demonstrated with a red FP fused with cytoplasmic beta-actin or tubulin-binding protein Tau34. In addition, heterotetramers appeared to be a unique model for biophysical characterization of Anthozoa FPs in pseudo-monomeric state.

  85. Chudakov D.M., Feofanov A.V., Mudrik N.N., Lukyanov S., Lukyanov K.A. (2003). Chromophore environment provides clue to "kindling fluorescent protein" riddle. J. Biol. Chem. 278 (9), 7215–9 [+]

    asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

  86. Chudakov D.M., Belousov V.V., Zaraisky A.G., Novoselov V.V., Staroverov D.B., Zorov D.B., Lukyanov S., Lukyanov K.A. (2003). Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21 (2), 191–4 [+]

    Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

  87. Fradkov A.F., Verkhusha V.V., Staroverov D.B., Bulina M.E., Yanushevich Y.G., Martynov V.I., Lukyanov S., Lukyanov K.A. (2002). Far-red fluorescent tag for protein labelling. Biochem. J. 368 (Pt 1), 17–21 [+]

    Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.

  88. Bulina M.E., Chudakov D.M., Mudrik N.N., Lukyanov K.A. (2002). Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis. BMC Biochem. 3, 7 [+]

    BACKGROUND: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. RESULTS: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. CONCLUSIONS: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

  89. Labas Y.A., Gurskaya N.G., Yanushevich Y.G., Fradkov A.F., Lukyanov K.A., Lukyanov S.A., Matz M.V. (2002). Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. U.S.A. 99 (7), 4256–61 [+]

    The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.

  90. Gurskaya N.G., Fradkov A.F., Terskikh A., Matz M.V., Labas Y.A., Martynov V.I., Yanushevich Y.G., Lukyanov K.A., Lukyanov S.A. (2001). GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507 (1), 16–20 [+]

    We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission lambda(max)=615-640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.

  91. Gurskaya N.G., Savitsky A.P., Yanushevich Y.G., Lukyanov S.A., Lukyanov K.A. (2001). Color transitions in coral's fluorescent proteins by site-directed mutagenesis. BMC Biochem. 2, 6 [+]

    Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters.

  92. Rebrikov D.V., Britanova O.V., Gurskaya N.G., Lukyanov K.A., Tarabykin V.S., Lukyanov S.A. (2000). Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization. Nucleic Acids Res. 28 (20), E90 [+]

    Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.

  93. Lukyanov K.A., Fradkov A.F., Gurskaya N.G., Matz M.V., Labas Y.A., Savitsky A.P., Markelov M.L., Zaraisky A.G., Zhao X., Fang Y., Tan W., Lukyanov S.A. (2000). Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275 (34), 25879–82 [+]

    It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

  94. Gurskaya N.G., Diatchenko L., Chenchik A., Siebert P.D., Khaspekov G.L., Lukyanov K.A., Vagner L.L., Ermolaeva O.D., Lukyanov S.A., Sverdlov E.D. (1996). Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal. Biochem. 240 (1), 90–7 [+]

    The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.

  95. Diatchenko L., Lau Y.F., Campbell A.P., Chenchik A., Moqadam F., Huang B., Lukyanov S., Lukyanov K., Gurskaya N., Sverdlov E.D., Siebert P.D. (1996). Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A. 93 (12), 6025–30 [+]

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.