Избранные публикации

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

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

   ID:1899
2. Dubovskii P.V., Dubinnyi M.A., Konshina A.G., Kazakova E.D., Sorokoumova G.M., Ilyasova T.M., Shulepko M.A., Chertkova R.V., Lyukmanova E.N., Dolgikh D.A., Arseniev A.S., Efremov R.G. (2017). Structural and Dynamic "Portraits" of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They? Biochemistry 56 (34), 4468–4477 [+]

   Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly (13)C- and (15)N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

   ID:1923
3. Chertkova R.V., Brazhe N.A., Bryantseva T.V., Nekrasov A.N., Dolgikh D.A., Yusipovich A.I., Sosnovtseva O., Maksimov G.V., Rubin A.B., Kirpichnikov M.P. (2017). New insight into the mechanism of mitochondrial cytochrome c function. PLoS ONE 12 (5), e0178280 [+]

   We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate-cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol-cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

   ID:1979
4. Pletneva N.V., Pletnev S., Pakhomov A.A., Chertkova R.V., Martynov V.I., Muslinkina L., Dauter Z., Pletnev V.Z. (2016). Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms. Acta Crystallogr D Struct Biol 72 (Pt 8), 922–32 [+]

   The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

   ID:1587
5. Kim Y.V., Gasparian M.E., Bocharov E.V., Chertkova R.V., Tkach E.N., Dolgikh D.A., Kirpichnikov M.P. (2015). New strategy for high-level expression and purification of biologically active monomeric TGF-β1/C77S in Escherichia coli. Mol. Biotechnol. 57 (2), 160–71 [+]

   Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

   ID:1682
6. Amdursky N., Ferber D., Bortolotti C.A., Dolgikh D.A., Chertkova R.V., Pecht I., Sheves M., Cahen D. (2014). Solid-state electron transport via cytochrome c depends on electronic coupling to electrodes and across the protein. Proc. Natl. Acad. Sci. U.S.A. 111 (15), 5556–61 [+]

   Electronic coupling to electrodes, Γ, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron-mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and ∼130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases Γ, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, Ea, of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower Ea values (from high-temperature data) and higher conductance at low temperatures (in the temperature-independent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width).

   ID:1664
7. Pepelina T.Y., Chertkova R.V., Ostroverkhova T.V., Dolgikh D.A., Kirpichnikov M.P., Grivennikova V.G., Vinogradov A.D. (2009). Site-directed mutagenesis of cytochrome c: reactions with respiratory chain components and superoxide radical. Biochemistry Mosc. 74 (6), 625–32 [+]

   Three forms of horse heart cytochrome c with specific substitutions of heme cleft surface located amino acid residues involved in specific interactions with ubiquinol:cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) were constructed, and their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The proteins with six (K27E/E69K/K72E/K86E/K87E/E90K and K8E/E62K/E69K/K72E/K86E/K87E) and eight (K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K) substitutions were inactive in the cytochrome c oxidase reaction, and their reduction rates by complex III were significantly lower than that seen with acetylated cytochrome c. The reduction of these modified cytochromes c under conditions where complex I generates superoxide was almost completely (about 90%) inhibited by superoxide dismutase. The genetically modified cytochromes c are useful analytical reagents for studies on superoxide generation by the mitochondrial respiratory chain. Quantitative comparison of superoxide-mediated cytochrome c reduction with hydrogen peroxide-mediated Amplex Red oxidation suggests that complex I within its native environment (submitochondrial particles) produces both superoxide (~50%) and hydrogen peroxide (~50%).

   ID:1684
8. Chertkova R.V., Kostanian I.A., Astapova M.V., Surina E.A., Dolgikh D.A., Kirpichnikov M.P. (2009). [An artificial protein, possessing biological activity of HL-60 human promyelocytic leukemia differentiation factor]. Bioorg. Khim. 29 (1), 30–7 [+]

   The ABB-df artificial protein was prepared by inserting the TGENHR biologically active peptide corresponding to the 41-46 sequence of the differentiation factor for the HL-60 cell line of the human promyelocyte leukemia into the N-terminus of the polypeptide chain of albebetin, an artificial protein with the preset structure. The ABB-df protein was found to induce the differentiation of HL-60 cells and to inhibit their proliferation; its efficiency was almost the same as that of the starting peptide. According to CD spectroscopy, the inclusion of the peptide fragment into albebetin exerts virtually no effect on the regular secondary structure of albebetin.

   ID:1681
9. Pepelina T.I.u., Chertkova R.V., Dolgikh D.A., Kirpichnikov M.P. (2009). [Role of individual lysine residues of horse cytochrome c in the formation of reactive complexes with components of the respiratory chain]. Bioorg. Khim. 36 (1), 98–104 [+]

   A number of mutant forms of horse cytochrome-c with single or double substitutions of lysine residues near the heme cavity was prepared that provided an interaction of mitochondrial ubiquinone with cytochrome-c reductase (EC 1.10.2.2) (complex III) and cytochrome-c oxidase (EC 1.9.3.1) (complex IV). The succinate cytochrome-c reductase and cytochrome-c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome-c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol-cytochrome-c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome-c oxidase.

   ID:1683
10. Chertkova R.V., Sharonov G.V., Feofanov A.V., Bocharova O.V., Latypov R.F., Chernyak B.V., Arseniev A.S., Dolgikh D.A., Kirpichnikov M.P. (2008). Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences. Mol. Cell. Biochem. 314 (1-2), 85–93 [+]

    Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) > h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.

    ID:1685
11. Bortolotti C.A., Borsari M., Sola M., Chertkova R., Dolgikh D., Kotlyar A., Facci P. (2007). Orientation-dependent kinetics of heterogeneous electron transfer for cytochrome c immobilized on gold: Electrochemical determination and theoretical prediction. Journal of Physical Chemistry C 111 (32), 12100–12105 [+]

    A systematic comparison between electron-transfer rate constants measured electrochemically for different cysteine-bearing mutants of cytochrome c chemisorbed on gold surfaces in different orientations has been performed. Experimental data have been correlated with electronic coupling theoretical estimates obtained from two different empirical models for the kinetics of protein electron transfer, the tunneling pathway model and the average packing density model. The results indicate that both models also hold in the case of immobilized redox proteins, outlining their role in the rational design of optimized electron-transfer-based bioinorganic interfaces.

    ID:1688
12. Lavrikova M.A., Zamotin V.V., Malisauskas M., Chertkova R.V., Kostanyan I.A., Dolgikh D.A., Kirpichnikov M.P., MorozovaRoche L.A. (2006). Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation. Biochemistry Mosc. 71 (3), 306–14 [+]

    The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.

    ID:1686
13. MorozovaRoche L.A., Zamotin V., Malisauskas M., Ohman A., Chertkova R., Lavrikova M.A., Kostanyan I.A., Dolgikh D.A., Kirpichnikov M.P. (2004). Fibrillation of carrier protein albebetin and its biologically active constructs. Multiple oligomeric intermediates and pathways. Biochemistry 43 (30), 9610–9 [+]

    We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-alpha(2) octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and beta-sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

    ID:1687