Laboratory of Biophotonics

Department of Genetics and Postgenomic Technologies

Head: Konstantin Lukyanov, corresponding member of the academy of sciences
kluk@ibch.ru+7(499)724-81-22

Fluorescent proteins, photoactivatable fluorescent proteins, genetically encoded fluorescent sensors, live cell labeling, genetically encoded photosensitizers

Laboratory works on development of novel fluorescent tags and methods of fluorescence labeling of biological objects. The main focus is on the fluorescent proteins of GFP family, and also on other methods of noninvasive visualization of structures and processes in live cells. These methods are widely used in biomedical research enabling deciphering the molecular mechanisms of various normal and pathological phenomena and facilitating preclinical drug screening.

We are fully equipped for gene-engineering works, mammalian cell culture works, fluorescence and laser scanning confocal microscopy, and optical spectroscopy.

Laboratory collaborates with many groups of the Institute: Total Synthesis Lab on studying physic-chemical properties of fluorescent protein chromophores and developing novel methods of fluorescence labeling using their analogs; Molecular technologies laboratory on development of novel fluorescent sensors; Laboratory of X-ray study on analysis of 3D structure of fluorescent proteins and structure-guided changes of their spectral properties; Laboratory of molecular bases of embryogenesis on application of fluorescent tools for developmental studies.

Also, Laboratory collaborates with Institute of Biomedical Technologies (Nizhny Novgorod Medical State Academy) on novel methods of super-resolution fluorescence microscopy and on fluorescence imaging of mouse tumor models; Laboratory of Physical Biochemistry (A.N. Bach Institute of Biochemistry) on fluorescence lifetime imaging microscopy of biological models; Kyril Solntsev (Georgia Institute of Technology, Atlanta, GA) on ultrafast spectroscopy of fluorescent proteins; laboratory of Anna Krylov (University of Southern California, Los Angeles, CA) on studying physic-chemical processes in fluorescent proteins; laboratory of Vladislav Verkhusha (Albert Einstein College of Medicine, Bronx, NY) on development of novel fluorescent proteins; laboratory of Jens Meiler (Vanderbilt University, Nashville, TN) on computer modeling of fluorescent proteins.

Laboratory was separated in 2009 from Laboratory of Molecular Technologies for Biology and Medicine headed by Sergey Lukyanov; our team is working with fluorescent proteins since 1999.

Novel fluorescent proteins

GFP and related fluorescent proteins are widely used as genetically encoded tags for visualization of proteins and cell populations in live systems. We use site-directed and random mutagenesis to create new variants of fluorescent proteins. This approach allows to generate proteins with usual spectral properties due to new chromophore structures or altered amino acid environment of the chromophore. In addition, we are working on improvement of fluorescent proteins characteristics such as brightness and photostability that are important for their practical applications.

Chudakov DM, et al. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010, 90, 1103-63.

Pletnev VZ, et al. 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. 2013, 69, 1850-60.

Sarkisyan KS, et al. Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime. Biophys J. 2015, 109, 380-9.

Mishin AS, et al. Novel uses of fluorescent proteins. Curr Opin Chem Biol. 2015, 27, 1-9.

Novel genetically encoded sensors

We are working on new fluorescent protein-based sensors for various regulatory activities, which enable quantitative visualization of the target events in live cells. For example, we recently developed ratiometric sensor for nonsense-mediated mRNA decay (NMD) and far-red sensor for caspase-3.

Gurskaya NG, et al. Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins. Nucleic Acids Res. 2012, 40, e57.

Pereverzev AP, et al. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep. 2015, 5, 7729.

Zlobovskaya OA, et al. Genetically encoded far-red fluorescent sensors for caspase-3 activity. Biotechniques. 2016, 60, 62-8.

Photoconversions of fluorescent proteins

Photoactivatable fluorescent proteins (PAFP) are used for tracking movements of proteins, organelles and cells in live systems, as well as for super-resolution fluorescence microscopy. Earlier, our team developed several PAFPs, such as KFP1, PS-CFP and Dendra, which were among the world's first members of this protein type. Currently, we continue to work on development of new PAFPs and PAFP-based techniques.

In 2009 we discovered oxidative photoconversion of green fluorescent proteins based on electron transfer from the chromophore to an external molecule of electron acceptor. We are studying mechanisms of this phenomenon and development of methods of its practical applications (for example, for enhancement of fluorescent protein photostability).

In collaboration with Nizhny Novgorod State Medical Academy we work on development of novel labels and methods of super-resolution fluorescence microscopy (PALM/STORM single molecule localization microscopy).

Chudakov DM, et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol. 2003, 21, 191-4.

Gurskaya NG, et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol. 2006, 24, 461-5.

Bogdanov AM, et al. Green fluorescent proteins are light-induced electron donors. Nat Chem Biol. 2009, 5, 459-61.

Bogdanov AM, et al. Cell culture medium affects GFP photostability: a solution. Nat Methods. 2009, 6, 859-60.

Mamontova AV, et al. Influence of cell growth conditions and medium composition on EGFP photostability in live cells. Biotechniques. 2015, 58, 258-61.

Genetically encoded photosensitizers

Phototoxic fluorescent proteins (the first such protein, KillerRed, was developed by us in 2006) produce reactive oxygen species (ROS) upon light illumination. We are developing this optoginetic technology, which makes it possible to induce oxidative stress in target cell compartments, inactivate proteins and kill specific cell populations using light.  

Bulina ME, et al. A genetically encoded photosensitizer. Nat Biotechnol. 2006, 24, 95-9.

Lukyanov KA, et al. Fluorescent proteins as light-inducible photochemical partners. Photochem Photobiol Sci. 2010, 9, 1301-6.

Serebrovskaya EO, et al. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed. J Biomed Opt. 2014,19, 071403.

Sarkisyan KS, et al. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS One. 2015, 10, e0145287.

2002—2006. A panel of photoactivated fluorescent proteins with different types of light-induced spectral transitions was introduced: nonflourescent-to-red (KFP1), blue-to-green (PS-CFP), and green-to-red (Dendra). These newly developed instruments KFP1, PS-CFP and Dendra were applied for precise photolabeling of cells, cell organelles, and proteins and subsequent tracking of the labeled object. The new tools were also used for monitoring the target protein degradation in an individual cell in real time using fluorescence confocal microscopy.

2005—2006. The first genetically encoded photosensitizer was created. This phototoxic red fluorescent protein named KillerRed can be used for precise light-induced destruction of proteins and cell killing.

2005—2007. A panel of improved fluorescent proteins for practical applications was created using the methods of directed molecular evolution. Particularly, there were obtained red and far-red fluorescent proteins, exceeding all known analogs in brightness. Bright far-red fluorescent proteins open up new prospects in whole-body fluorescent imaging technology.

2005—2008. First syntheses of chromophores of red fluorescent proteins (asFP595, Kaede, zFP538) and their structural analogs were performed. This work revealed various aspects of structure-properties relationship in this group of chromophores and allowed to propose promising amino-acid substitutions in fluorescent proteins to obtain variants with novel spectral properties.

Selected publications

  1. 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.

    ID:1602
  2. Acharya A., Bogdanov A.M., Grigorenko B.L., Bravaya K.B., Nemukhin A.V., Lukyanov K.A., Krylov A.I. (2016). Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem. Rev. , [+]

    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.

    ID:1666
  3. 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 [+]

    BACKGROUND:

    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.

    MATERIALS AND METHODS:

    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.

    RESULTS:

    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.

    CONCLUSION:

    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.

    ID:1601
  4. 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.

    ID:1555
  5. 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.

    ID:1529
  6. 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.

    ID:1526
  7. 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.

    ID:1407
  8. 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.

    ID:1373
  9. 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.

    ID:1374
  10. 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.

    ID:1419
  11. 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.

    ID:1527
  12. 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.

    ID:1528
  13. 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.

    ID:1416
  14. 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 [+]
    ID:1323
  15. 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.

    ID:1372
  16. 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.

    ID:1418
  17. 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.

    ID:1305
  18. 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
  19. 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.

    ID:1329
  20. 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.

    ID:1417
  21. 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.

    ID:1300
  22. Mishina N.M., Mishin A.S., Belyaev Y., Bogdanova E.A., Lukyanov S., Schultz C., Belousov V.V. (2015). Live-Cell STED Microscopy with Genetically Encoded Biosensor. Nano Lett. 15 (5), 2928–2932 [+]

    Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.

    ID:1259
  23. 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.

    ID:1247
  24. 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 [+]
    ID:1324
  25. 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.

    ID:1391
  26. GeorgeAbraham B., Sarkisyan K.S., Mishin A.S., Santala V., Tkachenko N.V., Karp M. (2015). Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements. PLoS ONE 10 (8), e0134436 [+]

    Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).

    ID:1406
  27. 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.

    ID:1554
  28. Baranov M.S., Solntsev K.M., Baleeva N.S., Mishin A.S., Lukyanov S.A., Lukyanov K.A., Yampolsky I.V. (2014). Red-shifted fluorescent aminated derivatives of a conformationally locked GFP chromophore. Chem. Eur. J. 20 (41), 13234–41 [+]

    A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red-shifted spectra, high fluorescence quantum yields and pH-independence in physiological pH range. The intra- and intermolecular mechanisms of radiationless deactivation of ABDI-BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with "infinite" photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.

    ID:1294
  29. Pletneva N.V., Pletnev S.V., Bogdanov A.M., Goriacheva E.A., Artemev I.V., Suslova E.A., Arkhipova S.F., Pletnev V.Z. (2014). Three dimensional structure of the dimeric gene-engineered variant of green fluorescent protein EGFP-K162Q in P6(1) crystal space group. Bioorg. Khim. 40 (4), 414–20 [+]
    ID:1326
  30. 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 [+]
    ID:1017
  31. 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.

    ID:1029
  32. 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.

    ID:908
  33. 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.

    ID:1032
  34. 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
  35. 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.

    ID:717
  36. 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.

    ID:831
  37. 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 [+]
    ID:1023
  38. 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.

    ID:513
  39. 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.

    ID:369
  40. 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.

    ID:363
  41. 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.

    ID:514
  42. 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.

    ID:22
  43. 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.

    ID:1037
  44. 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.

    ID:515
  45. 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.

    ID:516
  46. 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.

    ID:280
  47. 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.

    ID:517
  48. 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.

    ID:290

Konstantin Lukyanov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 34, office. 522
  • Phone: +7(499)724-81-22
  • E-mail: kluk@ibch.ru

Deep structure-functional analysis of amino acid substitutions on photophysical properties of green fluorescent proteins (2016-11-24)

A so called “fitness landscape” was for the first time experimentally probed at the whole protein level using GFP as a model. A unique approach developed in this work enabled to correlate a function (fluorescence) with amino acid sequence of several tens of thousands of random mutants, revealing a number of negative and positive epistatic interactions between substitutions. Characterization of the GFP fitness landscape allows for computer prediction of properties of new mutants of fluorescent proteins. It also has important implications for several fields including molecular evolution and protein design.

Using calculations of the possible electron transfer pathways from excited GFP chromophore to external molecules and further experimental verification of these hypotheses, we constructed mutants with blocked electron transfer pathway and correspondingly increased photostability. This strategy may represent a new approach toward enhancing photostability of fluorescent proteins.

 

Figure. (A) Scheme of GFP fitness landscape derived from analysis of 51000 mutants. The GFP sequence arranged in a circle, each column representing one amino acid site. In the first circle, the colour intensity of the squares indicates the brightness of a single mutation at the corresponding site relative to the wild type, shown in the centre. Sites with positive and negative epistatic interactions between pairs of mutations are connected by green and black lines, respectively. In circles further away from the centre, representing genotypes with multiple mutations, the fraction of the column coloured green (black) represents the fraction of genotypes corresponding to high (low) fluorescence among all assayed genotypes with a mutation at that site. (B) Electron transfer in GFP. Upper panel – scheme of calculated pathway of electron transfer from the chromophore to external acceptor molecule via tyrosine-145 as an intermediate electron acceptor. Bottom panel – photobleaching curves of EGFP and its mutants in the presence of oxidant in the medium, showing a dramatic enhancement of photostability due to blocking the electron transfer pathway. 

Publications

  1. Acharya A., Bogdanov A.M., Grigorenko B.L., Bravaya K.B., Nemukhin A.V., Lukyanov K.A., Krylov A.I. (2016). Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem. Rev. , [+]

    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.

    ID:1666
  2. 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.

    ID:1529
  3. 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.

    ID:1526

Method for analysis of nonsense-mediated mRNA decay in the single live cells using fluorescent proteins (2016-03-17)

Nonsense-mediated mRNA decay (NMD) is an evolutionary conserved mechanism of recognition and degradation of transcripts with a premature stop-codon. Recent studies demonstrated that NMD plays an important role in global regulation of gene expression. We developed novel reporter of NMD activity based on fluorescent proteins. It enables quantitative analysis of NMD activity at the level of single live cells (this cannot be done by any other known method of NMD analysis). Using our NMD reporter, we revealed strong differences of NMD activity between mammalian cell lines. Also, a phenomenon of significant heterogeneity of NMD activity within some cell lines was observed for the first time. In particular, subpopulations of cells with high and low NMD activity were detected in HEK293, Jurkat, and HaCaT cells. Our method opens new possibilities to decipher mechanisms of NMD regulation as well as to study consequences of low NMD activity on gene expression patterns and cell physiology.  

Publications

  1. 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.

    ID:1416
  2. 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
  3. 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.

    ID:1247