Laboratory of Genomics of Adaptive Immunity

Department of Genetics and Postgenomic Technologies

Head: Dmitriy Chudakov, D. Sc.
+7 (499) 742-81-22 ·

molecular tools, adaptive immunity, TCR repertoire, IG repertoire

The team works on development of molecular tools for the basic and biomedical studies, mainly in the filed of immunity and autoimmunity. The current focus of interest is the role of clonal T cell populations in the onset and development of autoimmune diseases, DNA technologies and software development for the quantitative analysis of individual TCR and antibody repertoires in health and disease.

Dmitry A. Bolotinj. r. f.
Olga V. Britanova, ph. d.s. r. f.
Evgen S. EgorovPhD stud.
Mark A. Izraelsont. q. - lab. as.
Sofya A. Kasatskayaj. r. f.
Vitalina I. Kirgizovastud.
Alexey A. Kotlobayj. r. f.
Mikhail . LebedinPhD stud.
Ekaterina M. Merzlyak, ph. d.
Ekaterina V. PutintsevaPhD stud.
Dmitriy S. Shcherbo, ph. d.r. f.
Irina I. Shemiakinaj. r. f.
Mikhail A. Shugayj. r. f.
Dmitry B. Staroverovr. f.
Maria A. Turchaninova, ph. d.r. f.
Olga I. Zuevaj. r. f.

Selected publications

  1. Bolotin D.A., Poslavsky S., Mitrophanov I., Shugay M., Mamedov I.Z., Putintseva E.V., Chudakov D.M. (2015). MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods 12 (5), 380–1 ID:1270
  2. Britanova O.V., Putintseva E.V., Shugay M., Merzlyak E.M., Turchaninova M.A., Staroverov D.B., Bolotin D.A., Lukyanov S., Bogdanova E.A., Mamedov I.Z., Lebedev Y.B., Chudakov D.M. (2014). Age-Related Decrease in TCR Repertoire Diversity Measured with Deep and Normalized Sequence Profiling. J. Immunol. 192 (6), 2689–98 [+]

    The decrease of TCR diversity with aging has never been studied by direct methods. In this study, we combined high-throughput Illumina sequencing with unique cDNA molecular identifier technology to achieve deep and precisely normalized profiling of TCR β repertoires in 39 healthy donors aged 6-90 y. We demonstrate that TCR β diversity per 10(6) T cells decreases roughly linearly with age, with significant reduction already apparent by age 40. The percentage of naive T cells showed a strong correlation with measured TCR diversity and decreased linearly up to age 70. Remarkably, the oldest group (average age 82 y) was characterized by a higher percentage of naive CD4(+) T cells, lower abundance of expanded clones, and increased TCR diversity compared with the previous age group (average age 62 y), suggesting the influence of age selection and association of these three related parameters with longevity. Interestingly, cross-analysis of individual TCR β repertoires revealed a set >10,000 of the most representative public TCR β clonotypes, whose abundance among the top 100,000 clones correlated with TCR diversity and decreased with aging.

  3. Linnemann C., Heemskerk B., Kvistborg P., Kluin R.J., Bolotin D.A., Chen X., Bresser K., Nieuwland M., Schotte R., Michels S., GomezEerland R., Jahn L., Hombrink P., Legrand N., Shu C.J., Mamedov I.Z., Velds A., Blank C.U., Haanen J.B., Turchaninova M.A., Kerkhoven R.M., Spits H., Hadrup S.R., Heemskerk M.H., Blankenstein T., Chudakov D.M., Bendle G.M., Schumacher T.N. (2013). High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat. Med. , [+]

    The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

  4. Bolotin D.A., Shugay M., Mamedov I.Z., Putintseva E.V., Turchaninova M.A., Zvyagin I.V., Britanova O.V., Chudakov D.M. (2013). MiTCR: software for T-cell receptor sequencing data analysis. Nat. Methods 10 (9), 813–4 ID:886
  5. 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 [+]
  6. Turchaninova M.A., Britanova O.V., Bolotin D.A., Shugay M., Putintseva E.V., Staroverov D.B., Sharonov G., Shcherbo D., Zvyagin I.V., Mamedov I.Z., Linnemann C., Schumacher T.N., Chudakov D.M. (2013). Pairing of T-cell receptor chains via emulsion PCR. European journal of immunology , [+]

    Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains - both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are co-expressed in the same living cells. This article is protected by copyright. All rights reserved.

  7. Bolotin D.A., Mamedov I.Z., Britanova O.V., Zvyagin I.V., Shagin D., Ustyugova S.V., Turchaninova M.A., Lukyanov S., Lebedev Y.B., Chudakov D.M. (2012). Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms. European journal of immunology , [+]

    The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of NGS applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

  8. Britanova O.V., Bochkova A.G., Staroverov D.B., Fedorenko D.A., Bolotin D.A., Mamedov I.Z., Turchaninova M.A., Putintseva E.V., Kotlobay A.A., Lukyanov S., Novik A.A., Lebedev Y.B., Chudakov D.M. (2012). First autologous hematopoietic SCT for ankylosing spondylitis: a case report and clues to understanding the therapy. Bone marrow transplantation , ID:732
  9. Mamedov I.Z., Britanova O.V., Bolotin D.A., Chkalina A.V., Staroverov D.B., Zvyagin I.V., Kotlobay A.A., Turchaninova M.A., Fedorenko D.A., Novik A.A., Sharonov G.V., Lukyanov S., Chudakov D.M., Lebedev Y.B. (2011). Quantitative tracking of T cell clones after haematopoietic stem cell transplantation. EMBO Mol Med 3 (4), 201–7 [+]

    Autologous haematopoietic stem cell transplantation is highly efficient for the treatment of systemic autoimmune diseases, but its consequences for the immune system remain poorly understood. Here, we describe an optimized RNA-based technology for unbiased amplification of T cell receptor beta-chain libraries and use it to perform the first detailed, quantitative tracking of T cell clones during 10 months after transplantation. We show that multiple clones survive the procedure, contribute to the immune response to activated infections, and form a new skewed and stable T cell receptor repertoire.

  10. Luker K.E., Mihalko L.A., Schmidt B.T., Lewin S.A., Ray P., Shcherbo D., Chudakov D.M., Luker G.D. (2011). In vivo imaging of ligand receptor binding with Gaussia luciferase complementation. Nat. Med. 18 (1), 172–7 [+]

    Studies of ligand-receptor binding and the development of receptor antagonists would benefit greatly from imaging techniques that translate directly from cell-based assays to living animals. We used Gaussia luciferase protein fragment complementation to quantify the binding of chemokine (C-X-C motif) ligand 12 (CXCL12) to chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. Studies established that small-molecule inhibitors of CXCR4 or CXCR7 specifically blocked CXCL12 binding in cell-based assays and revealed differences in kinetics of inhibiting chemokine binding to each receptor. Bioluminescence imaging showed CXCL12-CXCR7 binding in primary and metastatic tumors in a mouse model of breast cancer. We used this imaging technique to quantify drug-mediated inhibition of CXCL12-CXCR4 binding in living mice. We expect this imaging technology to advance research in areas such as ligand-receptor interactions and the development of new therapeutic agents in cell-based assays and small animals.

  11. 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.

  12. Zvyagin I.V., Mamedov I.Z., Britanova O.V., Staroverov D.B., Nasonov E.L., Bochkova A.G., Chkalina A.V., Kotlobay A.A., Korostin D.O., Rebrikov D.V., Lukyanov S., Lebedev Y.B., Chudakov D.M. (2010). Contribution of functional KIR3DL1 to ankylosing spondylitis. Cellular & molecular immunology , [+]

    Increasing evidence points to a role for killer immunoglobulin-like receptors (KIRs) in the development of autoimmune diseases. In particular, a positive association of KIR3DS1 (activating receptor) and a negative association of KIR3DL1 (inhibitory receptor) alleles with ankylosing spondylitis (AS) have been reported by several groups. However, none of the studies analyzed these associations in the context of functionality of polymorphic KIR3DL1. To better understand how the KIR3DL1/3DS1 genes determine susceptibility to AS, we analyzed the frequencies of alleles and genotypes encoding functional (KIR3DL1*F) and non-functional (KIR3DL1*004) receptors. We genotyped 83 AS patients and 107 human leukocyte antigen (HLA)-B27-positive healthy controls from the Russian Caucasian population using a two-stage sequence-specific primer PCR, which distinguishes KIR3DS1, KIR3DL1*F and KIR3DL1*004 alleles. For the patients carrying two functional KIR3DL1 alleles, those alleles were additionally genotyped to identify KIR3DL1*005 and KIR3DL1*007 alleles, which are functional but are expressed at low levels. KIR3DL1 was negatively associated with AS at the expense of KIR3DL1*F but not of KIR3DL1*004. This finding indicates that the inhibitory KIR3DL1 receptor protects against the development of AS and is not simply a passive counterpart of the segregating KIR3DS1 allele encoding the activating receptor. However, analysis of genotype frequencies indicates that the presence of KIR3DS1 is a more important factor for AS susceptibility than the absence of KIR3DL1*F. The activation of either natural killer (NK) or T cells via the KIR3DS1 receptor can be one of the critical events in AS development, while the presence of the functional KIR3DL1 receptor has a protective effect. Nevertheless, even individuals with a genotype that carried two inhibitory KIR3DL1 alleles expressed at high levels could develop AS.Cellular & Molecular Immunology advance online publication, 6 September 2010; doi:10.1038/cmi.2010.42.

  13. 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.

  14. 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
  15. Mamedov I.Z., Britanova O.V., Chkalina A.V., Staroverov D.B., Amosova A.L., Mishin A.S., Kurnikova M.A., Zvyagin I.V., Mutovina Z.Y., Gordeev A.V., Khaidukov S.V., Sharonov G.V., Shagin D.A., Chudakov D.M., Lebedev Y.B. (2009). Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients. Autoimmunity 42 (6), 525–36 [+]

    Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

  16. 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.

  17. 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.

  18. Shcherbo D., Murphy C.S., Ermakova G.V., Solovieva E.A., Chepurnykh T.V., Shcheglov A.S., Verkhusha V.V., Pletnev V.Z., Hazelwood K.L., Roche P.M., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M. (2009). Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418 (3), 567–74 [+]

    A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

  19. Zhang L., Gurskaya N.G., Kopantseva Y.E., Mudrik N.N., Vagner L.L., Lukyanov K.A., Chudakov D.M. (2009). [Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP protein]. Bioorg. Khim. 36 (2), 187–92 [+]

    The site-directed mutagenesis of the monomeric red fluorescent protein TagRFP and its variants was performed with the goal of generating reversibly photoactivatable fluorescent proteins. Amino acids at positions 69, 148, 165, 179, and 181 (enumeration according to the green fluorescent protein GFP) were shown to play a key role in the manifestation of the photoactivatable properties. A reversibly photoactivatable red fluorescent protein KFP-HC with excitation and emission maxima at 585 and 615 nm, respectively, was generated. The KFP-HC fluorescent intensity was decreased by 5-10 times under green light (530-560 nm) irradiation (due to the fall of the fluorescence quantum yield) and restored under irradiation with blue light (450-490 nm) or after incubation in the dark (time of half reconstruction of 30 min).

  20. 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.

  21. 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 [+]

    A novel fluorescent protein Katushka with far-red emission preferable for signal registration inside animal tissues was created. Katushka is 10 fold brighter than other far-red proteins and is also characterized with fast maturation, high pH-stability and photostability. This constellation of properties makes it an instrument of choice for in vivo labeling of particular cells within whole organisms. A monomeric variant of Katushka named mKate was introduced for intracellular protein localization studies.

  22. 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.

  23. 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.

  24. 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.

  25. 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.

  26. 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.

  27. 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.

  28. 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.

  29. 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.

  30. 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.

  31. 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.


Head of the laboratory

Dmitriy Chudakov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 34, office 522
  • Phone: +7 (499) 742-81-22
  • E-mail: