Мартынов Владимир Иванович

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
1970–1975 Россия, Москва Московский государственный университет им. М.В. Ломоносова (химический факультет) Диплом химика
1983 Россия, Москва Московский государственный университет им. М.В. Ломоносова (химический факультет) Диплом кандидата химических наук

Научные интересы

Основным направлением его научных работ является изучение структуры и функции светочувствительных белков.

Основные научные результаты

В 1983 г. им было впервые показано, что зрительный родопсин, один из членов обширного семейства G-белок связанных рецепторов, состоит из семи трансмембранных доменов, пронизывающих фоторецепторную мембрану (Martynov et al., 1983). Сейчас это семейство так и называется, 7-ми-доменные трансмембранные рецепторы (7TMD receptors). В. им была защищена кандидатская диссертация на тему «Изучение молекулярной организации зрительного родопсина в фоторецепторной мембране». В настоящее время его исследования сосредоточены на флуоресцентных белках семейства GFP и посвящены изучению структуры и ее влиянию на биохимические, оптические и функциональные свойства этих белков.

Избранные публикации

  1. 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
  2. Pletneva N.V., Pletnev V.Z., Souslova E., Chudakov D.M., Lukyanov S., Martynov V.I., Arhipova S., Artemyev I., Wlodawer A., Dauter Z., Pletnev S. (2013). Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 6), 1005–12 [+]

    The yellow fluorescent protein phiYFPv (λem(max) ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow-orange range (535-555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The `yellow' chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

    ID:850
  3. Pakhomov A.A., Martynov V.I. (2011). Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp. Biochem. Biophys. Res. Commun. 407 (1), 230–5 [+]

    Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λ(em)=537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling - mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λ(em)=508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore.

    ID:823
  4. Pletneva N.V., Pletnev V.Z., Lukyanov K.A., Gurskaya N.G., Goryacheva E.A., Martynov V.I., Wlodawer A., Dauter Z., Pletnev S. (2010). Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis. J. Biol. Chem. 285 (21), 15978–84 [+]

    The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (lambda(ex) = 480 nm, lambda(em) = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C(alpha) and C(beta) atoms in the sp(3) hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (lambda = 250-300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C(alpha)=C(beta) bond and the conjugated system of pi-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.

    ID:404
  5. Pletnev S., Gurskaya N.G., Pletneva N.V., Lukyanov K.A., Chudakov D.M., Martynov V.I., Popov V.O., Kovalchuk M.V., Wlodawer A., Dauter Z., Pletnev V. (2009). Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J. Biol. Chem. 284 (46), 32028–39 [+]

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

    ID:299
  6. Pakhomov A.A., Martynov V.I. (2009). Posttranslational chemistry of proteins of the GFP family. Biochemistry Mosc. 74 (3), 250–9 [+]This review focuses on the current knowledge about posttranslational chemistry underlying the diverse optical properties of GFP-like proteins. ID:822
  7. Pletneva N.V., Pletnev S.V., Chudakov D.M., Tikhonova T.V., Popov V.O., Martynov V.I., Wlodawer A., Dauter Z., Pletnev V.Z. (2009). [Three-dimensional structure of yellow fluorescent protein zYFP538 from Zoanthus sp. at the resolution 1.8 angstrom]. Bioorg. Khim. 33 (4), 421–30 [+]

    The three-dimensional structure of yellow fluorescent proteins zYFP538 (zFP538) from the button polyp Zoanthus sp. was determined at a resolution of 1.8 angstrom by X-ray analysis. The monomer of zYFP538 adopts a structure characteristic of the green fluorescent protein (GFP) family, a beta-barrel formed from 11 antiparallel beta segments and one internal alpha helix with a chromophore embedded into it. Like the TurboGFP, the beta-barrel of zYFP538 contains a water-filled pore leading to the chromophore Tyr67 residue, which presumably provides access of molecular oxygen necessary for the maturation process. The post-translational modification of the chromophore-forming triad Lys66-Tyr67-Gly68 results in a tricyclic structure consisting of a five-membered imidazolinone ring, a phenol ring of the Tyr67 residue, and an additional six-membered tetrahydropyridine ring. The chromophore formation is completed by cleavage of the protein backbone at the Calpha-N bond of Lys66. It was suggested that the energy conflict between the buried positive charge of the intact Lys66 side chain in the hydrophobic pocket formed by the Ile44, Leu46, Phe65, Leu204 and Leu219 side chains is the most probable trigger that induces the transformation of the bicyclic green form to the tricyclic yellow form. A stereochemical analysis of the contacting surfaces at the intratetramer interfaces helped reveal a group of conserved key residues responsible for the oligomerization. Along with others, these residues should be taken into account in designing monomeric forms suitable for practical application as markers of proteins and cell organelles.

    ID:306
  8. Pakhomov A.A., Tretiakova Iu.A., Martynov V.I. (2009). [Posttranslational reactions resulting in a long-wavelength shift in the spectra of asFP595 protein from Anemonia sulcata]. Bioorg. Khim. 36 (1), 117–21 [+]

    In most fluorescent proteins characterized by light absorption and emission in the red and the far-red spectral region (550-650 nm), the chromophore pi system is extended at the expense of the additional oxidation of the GFP-like structure and the formation of an acylimine substituent. As distinct from these proteins, the photoactivateable protein asFP595 contains a chromophore with the keto group substituted for an acylimine substituent. In this work, we studied the reactions that result in a bathochromic shift in the spectrum of asFP595. Maturation kinetics analysis has shown the generation of the immature form containing a protonated chromophore (absorption at 420 nm) at the intermediate step, as in the case of other red fluorescent proteins, which then is isobestically converted into the final mature form (568 nm). Mass spectrometric analysis of the chromopeptide isolated from immature asFP595 has demonstrated that the intermediate form contains a GFP-type chromophore. It has also been found that the oxidation of the GFP chromophore is accompanied by the generation of an equimolar amount of hydro gen peroxide. The intermediate products of oxidation have been analyzed by the mutagenesis of the first chromophore-generating amino acid residue. It has been demonstrated that in the case of all mutants studied, chromophore synthesis does not terminate at the stage of the acylimine derivative, but immediately results in the fragmentation of the main chain of the protein and in the formation of the keto form.

    ID:825
  9. Pakhomov A.A., Tretyakova S.A., Martynov V.I. (2009). The influence of chromophore-protein interactions on spectroscopic properties of the yellow fluorescent protein. Dokl. Biochem. Biophys. 445, 207–9 ID:828
  10. Pakhomov A.A., Martynov V.I. (2009). [3D-structure determination of fluorescent proteins by homology modeling combined with mass spectrometry]. Bioorg. Khim. 37 (3), 429–32 [+]

    A method for the 3D-structure generation of GFP-like fluorescent proteins is presented. The method is based on a combination of homology modeling for the overall spatial structure determination and mass spectrometry for the chromophore structure identification. The proposed approach can be applied to the spatial structure determination ofnoncrystalizable GFP homologs.

    ID:829
  11. Martynov V.I., Pakhomov A.A., Popova N.V., Deyev I.E., Petrenko A.G. (2009). Synthetic Fluorophores for Visualizing Biomolecules in Living Systems. Acta Naturae 8 (4), 33–46 [+]

    The last decade has witnessed significant advance in the imaging of living systems using fluorescent markers. This progress has been primarily associated with the discovery of different spectral variants of fluorescent proteins. However, the fluorescent protein technology has its own limitations and, in some cases, the use of low-molecular-weight fluorophores is preferable. In this review, we describe the arsenal of synthetic fluorescent tools that are currently in researchers' hands and span virtually the entire spectrum, from the UV to visible and, further, to the near-infrared region. An overview of recent advances in site-directed introduction of synthetic fluorophores into target cellular objects is provided. Application of these fluorescent probes to the solution of a wide range of biological problems, in particular, to the determination of local ion concentrations and pH in living systems, is discussed.

    ID:1697
  12. Pakhomov A.A., Martynov V.I. (2008). GFP family: structural insights into spectral tuning. Chem. Biol. 15 (8), 755–64 [+]

    В обзоре систематизированы белки семейства GFP согласно посттрансляционным реакциям и структурам хромофоров. Обобщены современные данные, касающиеся влияния структуры на спектральные свойства этих белков.

    ID:62
  13. Pakhomov A.A., Martynov V.I. (2007). Chromophore aspartate oxidation-decarboxylation in the green-to-red conversion of a fluorescent protein from Zoanthus sp. 2. Biochemistry 46 (41), 11528–35 [+]

    В статье описана новая посттрансляционная реакция белков семейства GFP, которая лежит в основе красной флуоресценции z2FP574 из Zoanthus sp. 2.

    ID:63
  14. Pletneva N., Pletnev V., Tikhonova T., Pakhomov A.A., Popov V., Martynov V.I., Wlodawer A., Dauter Z., Pletnev S. (2007). Refined crystal structures of red and green fluorescent proteins from the button polyp Zoanthus. Acta Crystallogr. D Biol. Crystallogr. 63 (Pt 10), 1082–93 [+]

    Определена кристаллическая структура белка z2FP574 и его мутантов на различных стадиях созревания. Сделаны выводы, касающиеся структурных основ зелено-красного превращения флуоресценции z2FP574 из Zoanthus sp. 2.

    ID:64
  15. Tretyakova Y.A., Pakhomov A.A., Martynov V.I. (2007). Chromophore structure of the kindling fluorescent protein asFP595 from Anemonia sulcata. J. Am. Chem. Soc. 129 (25), 7748–9 [+]

    С помощью масс-спектрометрии высокого разрешения показано, что хромофор белка asFP595 является посттрансляционным продуктом реакции гидролиза DsRed-подобного хромофора и представляет собой кето-производное.

    ID:66
  16. Pakhomov A.A., Pletneva N.V., Balashova T.A., Martynov V.I. (2006). Structure and reactivity of the chromophore of a GFP-like chromoprotein from Condylactis gigantea. Biochemistry 45 (23), 7256–64 [+]

    Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and 1H and 13C NMR of the chromopeptide reveal that the protein contains a chromophore with a chemical structure identical to that of the red fluorescent protein from Discosoma sp. A single A63G substitution demonstrates that the nature of the first amino acid of the XYG chromophore-forming sequence is dispensable for the chromoprotein red shift development. It has been recently proposed that post-translational reactions at the acylimine, a chemical group that accounts for the red fluorescence, might be an additional source of spectral diversity of proteins homologous to the Aequorea victoria green fluorescent protein (GFP). We have examined the reactivity of the chromophore acylimine group within the C. gigantea purple chromoprotein. Like other proteins with the acylimine-modified chromophore, the purple chromoprotein suffers a hypsochromic spectral shift to the GFP-like absorbance (386 nm) upon mild denaturation. NMR analysis of the chromopeptide suggests this hypsochromic spectral shift is due to H2O addition across the C=N bond of the acylimine. However, unlike the red fluorescent protein from Discosoma sp., denatured under harsh conditions, the wild-type chromoprotein exhibits only slight fragmentation, which is induced by complete hydrolysis of the acylimine. A model suggesting the influence of the amino acid X side chain on protein fragmentation is presented.

    ID:821
  17. Pletneva N., Pletnev S., Tikhonova T., Popov V., Martynov V., Pletnev V. (2006). Structure of a red fluorescent protein from Zoanthus, zRFP574, reveals a novel chromophore. Acta Crystallogr. D Biol. Crystallogr. 62 (Pt 5), 527–32 [+]

    The three-dimensional structure of the red fluorescent protein (RFP) zRFP574 from the button polyp Zoanthus sp. (two dimers per asymmetric unit, 231 x 4 amino acids) has been determined at 2.4 A resolution in space group C222(1). The crystal structure, refined to a crystallographic R factor of 0.203 (R(free) = 0.249), adopts the beta-barrel fold composed of 11 strands similar to that of the yellow fluorescent protein zYFP538. The zRFP574 chromophore, originating from the protein sequence Asp66-Tyr67-Gly68, has a two-ring structure typical of GFP-like proteins. The bond geometry of residue 66 shows the strong tendency of the corresponding C(alpha) atom to sp(2) hybridization as a consequence of N-acylimine bond formation. The zRFP574 chromophore contains the 65-66 cis-peptide bond characteristic of red fluorescent proteins. The chromophore phenolic ring adopts a cis conformation coplanar with the imidazolinone ring. The crystallographic study has revealed an unexpected chemical feature of the internal chromophore. A decarboxylated side chain of the chromophore-forming residue Asp66 has been observed in the structure. This additional post-translational modification is likely to play a key role in the bathochromic shift of the zRFP574 spectrum.

    ID:824
  18. 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
  19. Pakhomov A.A., Martynova N.Y., Gurskaya N.G., Balashova T.A., Martynov V.I. (2004). Photoconversion of the chromophore of a fluorescent protein from Dendronephthya sp. Biochemistry Mosc. 69 (8), 901–8 [+]

    A green fluorescent protein from the coral Dendronephthya sp. (Dend FP) is characterized by an irreversible light-dependent conversion to a red-emitting form. The molecular basis of this phenomenon was studied in the present work. Upon UV-irradiation at 366 nm, the absorption maximum of the protein shifted from 494 nm (the green form) to 557 nm (the red form). Concurrently, in the fluorescence spectra the emission maximum shifted from 508 to 575 nm. The green form of native Dend FP was shown to be a dimer, and the oligomerization state of the protein did not change during its conversion to the red form. By contrast, UV-irradiation caused significant intramolecular changes. Unlike the green form, which migrates in SDS-polyacrylamide gels as a single band corresponding to a full-length 28-kD protein, the red form of Dend FP migrated as two fragments of 18- and 10-kD. To determine the chemical basis of these events, the denatured red form of Dend FP was subjected to proteolysis with trypsin. From the resulting hydrolyzate, a chromophore-containing peptide was isolated by HPLC. The structure of the chromophore from the Dend FP red form was established by methods of ESI, tandem mass spectrometry (ESI/MS/MS), and NMR-spectroscopy. The findings suggest that the light-dependent conversion of Dend FP is caused by generation of an additional double bond in the side chain of His65 and a resulting extension of the conjugated system of the green form chromophore. Thus, classified by the chromophore structure, Dend FP should be referred to the Kaede subfamily of GFP-like proteins.

    ID:820
  20. Martynov V.I., Maksimov B.I., Martynova N.Y., Pakhomov A.A., Gurskaya N.G., Lukyanov S.A. (2003). A purple-blue chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins. J. Biol. Chem. 278 (47), 46288–92 [+]

    A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).

    ID:819
  21. Fradkov A.F., Verkhusha V.V., Staroverov D.B., Bulina M.E., Yanushevich Y.G., Martynov V.I., Lukyanov S., Lukyanov K.A. (2002). Far-red fluorescent tag for protein labelling. Biochem. J. 368 (Pt 1), 17–21 [+]

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

    ID:826
  22. Gurskaya N.G., Fradkov A.F., Terskikh A., Matz M.V., Labas Y.A., Martynov V.I., Yanushevich Y.G., Lukyanov K.A., Lukyanov S.A. (2001). GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507 (1), 16–20 [+]

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

    ID:827
  23. Martynov V.I., Savitsky A.P., Martynova N.Y., Savitsky P.A., Lukyanov K.A., Lukyanov S.A. (2001). Alternative cyclization in GFP-like proteins family. The formation and structure of the chromophore of a purple chromoprotein from Anemonia sulcata. J. Biol. Chem. 276 (24), 21012–6 [+]

    Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.

    ID:818
  24. Martynov V.I., Kostina M.B., Feigina M.I.u., Miroshnikov A.I. (1983). Limited proteolysis studies on molecular organization of bovine rhodopsin in the photoreceptor membrane. Bioorg Khim. 9, 734–745 [+]

    С помощью ограниченного протеинолиза на диск-мембранах нативной и инвертированной ориентации впервые показано, что зрительный родопсин состоит из семи трансмембранных доменов, пронизывающих фоторецепторную мембрану. Определены участки полипептидной цепи, экспонированные в цитоплазматическое и внутридисковое пространство фоторецепторной клетки, а также участки, погруженные в фосфолипидный матрикс.

    ID:65