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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Natashin P.V., Ding W..., Eremeeva E.V., Markova S.V., Lee J..., Vysotski E.S., Liu Z.J.
Заглавие : Structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction
Колич.характеристики :13 с
Коллективы : RFBR [12-04-91153, 12-04-00131, 14-04-31092]; Chinese Academy of Sciences; NSFC; Programs of the Government of the Russian Federation 'Measures to Attract Leading Scientists to Russian Educational Institutions' [11.G34.31.0058]; RAS; Russian Federation 'Leading Science School' [3951.2012.4]
Место публикации : Acta Crystallogr. Sect. D-Biol. Crystallogr.: WILEY-BLACKWELL, 2014. - Vol. 70. - С. 720-732. - ISSN 0907-4449, DOI 10.1107/S1399004713032434. - ISSN 1399-0047
Примечания : Cited References: 71. - We acknowledge the use of beamline BL17U1 at the Shanghai Synchrotron Radiation Facility, China. This work was supported by RFBR grants 12-04-91153, 12-04-00131 and the China-Russia International Collaboration grant from the Chinese Academy of Sciences and NSFC, by the Programs of the Government of the Russian Federation 'Measures to Attract Leading Scientists to Russian Educational Institutions' (grant 11.G34.31.0058) and 'Molecular and Cellular Biology' of the RAS, the President of the Russian Federation 'Leading Science School' (grant 3951.2012.4). PVN and EVE were supported by RFBR grant 14-04-31092.
Предметные рубрики: AEQUORIN BIOLUMINESCENCE
SEQUENCE-ANALYSIS
CRYSTAL-STRUCTURE
CA2+-BINDING PHOTOPROTEIN
VIOLET BIOLUMINESCENCE
CALCIUM CONCENTRATION
ANGSTROM RESOLUTION
RECOMBINANT OBELIN
MNEMIOPSIS-LEIDYI
EXCITED-STATES
Аннотация: Ca2+-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca2+ inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. Here, the crystal structures of the Y138F obelin mutant before and after bioluminescence are reported at 1.72 and 1.30 angstrom resolution, respectively. The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild-type obelin gives clear evidence that the substitution of Tyr by Phe does not affect the overall structure of both Y138F obelin and its product following Ca2+ discharge compared with the corresponding conformational states of wild-type obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild-type obelins, there is a substantial difference: in the cavity of Y138F obelin a water molecule corresponding to W2 in wild-type obelin is not found. However, in Ca2+-discharged Y138F obelin this water molecule now appears in the same location. This finding, together with the observed much slower kinetics of Y138F obelin, clearly supports the hypothesis that the function of a water molecule in this location is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion before its decomposition into the excited-state product. Although obelin differs from other hydromedusan Ca2+-regulated photoproteins in some of its properties, they are believed to share a common mechanism.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Stepanyuk G.A., Golz S..., Markova S.V., Frank L.A., Lee J..., Vysotski E.S.
Заглавие : Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein
Колич.характеристики :7 с
Место публикации : FEBS Lett.: ELSEVIER SCIENCE BV, 2005. - Vol. 579, Is. 5. - С. 1008-1014. - ISSN 0014-5793, DOI 10.1016/j.febslet.2005.01.004
Примечания : Cited References: 49
Предметные рубрики: FIREFLY LUCIFERASE
SEQUENCE-ANALYSIS
CA2+-REGULATED PHOTOPROTEINS
CA2+-DISCHARGED PHOTOPROTEIN
VIOLET BIOLUMINESCENCE
INTRACELLULAR CALCIUM
ENDOPLASMIC-RETICULUM
ANGSTROM RESOLUTION
CRYSTAL-STRUCTURE
APOAEQUORIN CDNA
Ключевые слова (''Своб.индексиров.''): coelenterazine--calcium--reporter protein--mammalian expression--fluorescence spectrum
Аннотация: The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambda(max) = 469 nm) and obelin (lambda(max) = 482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having lambda(max) = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to lambda(max) = 501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection. (C) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva E.V., Markova S.V., van Berkel WJH, Vysotski E.S.
Заглавие : Role of key residues of obelin in coelenterazine binding and conversion into 2-hydroperoxy adduct
Колич.характеристики :7 с
Коллективы : RFBR [12-04-00131]; Programs of the Government of Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" [11.G34.31.0058]; "Molecular and Cellular Biology" of RAS, President of Russian Federation "Leading science school" [3951.2012.4]; Wageningen University Sandwich PhD-Fellowship Program
Место публикации : J. Photochem. Photobiol. B-Biol.: ELSEVIER SCIENCE SA, 2013. - Vol. 127. - С. 133-139. - ISSN 1011-1344, DOI 10.1016/j.jphotobiol.2013.08.012
Примечания : Cited References: 65. - The work was supported by RFBR grant 12-04-00131, by the Programs of the Government of Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.0058), "Molecular and Cellular Biology" of RAS, President of Russian Federation "Leading science school" (grant 3951.2012.4). E.V.E. was supported by Wageningen University Sandwich PhD-Fellowship Program.
Предметные рубрики: CA2+-REGULATED PHOTOPROTEINS
SEQUENCE-ANALYSIS
CRYSTAL-STRUCTURE
APO-OBELIN
CA2+-BINDING PHOTOPROTEIN
VIOLET BIOLUMINESCENCE
AEQUORIN REGENERATION
ANGSTROM RESOLUTION
RECOMBINANT OBELIN
MNEMIOPSIS-LEIDYI
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--obelin--aequorin--photoprotein
Аннотация: Bioluminescence of a variety of marine organisms is caused by monomeric Ca2+-regulated photoproteins, to which a peroxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxycoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, coelenterazine binding, and kinetics of active obelin formation. We demonstrate that Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation; substitution of any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond network formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of coelenterazine in the active site and subsequent stabilization of the 2-hydroperoxy adduct of coelenterazine. His175 might serve as a proton shuttle during 2-hydroperoxycoelenterazine formation. (C) 2013 Elsevier B.V. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Malikova N.P., Burakova L.P., Markova S.V., Vysotski E.S.
Заглавие : Characterization of hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca(2+)concentration
Колич.характеристики :12 с
Коллективы : RFBR [12-04-00131]; Government of the Russian Federation [11.G34.31.0058]; Russian Academy of Sciences; Russian Federation "Leading Science School" [3951.2012.4]
Место публикации : Anal. Bioanal. Chem.: SPRINGER HEIDELBERG, 2014. - Vol. 406, Is. 23. - С. 5715-5726. - ISSN 1618-2642, DOI 10.1007/s00216-014-7986-2. - ISSN 1618-2650
Примечания : Cited References: 67. - This work was supported by RFBR grant 12-04-00131, by the programs of the Government of the Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.0058) and "Molecular and Cellular Biology" of the Russian Academy of Sciences, and the grant from the President of the Russian Federation "Leading Science School" (3951.2012.4).
Предметные рубрики: LIGHT-SENSITIVE PHOTOPROTEIN
CTENOPHORE BEROE ABYSSICOLA
GREEN-FLUORESCENT PROTEIN
INTRACELLULAR CALCIUM
SEQUENCE-ANALYSIS
CA-2+-ACTIVATED PHOTOPROTEIN
CA2+-BINDING PHOTOPROTEIN
SEMISYNTHETIC AEQUORINS
LUMINESCENT PROTEIN
RECOMBINANT OBELIN
Ключевые слова (''Своб.индексиров.''): calcium--coelenterazine--aequorin--obelin--clytin--mitrocomin
Аннотация: Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca2+-regulated photoproteins have been successfully used for this purpose for many years. Here we report the results of comparative studies on the properties of recombinant aequorin from Aequorea victoria, recombinant obelins from Obelia geniculata and Obelia longissima, recombinant mitrocomin from Mitrocoma cellularia, and recombinant clytin from Clytia gregaria as intracellular calcium indicators in a set of identical in vitro and in vivo experiments. Although photoproteins reveal a high degree of identity of amino acid sequences and spatial structures, and, apparently, have a common mechanism for the bioluminescence reaction, they were found to differ in the Ca2+ concentration detection limit, the sensitivity of bioluminescence to Mg2+, and the rates of the rise of the luminescence signal with a sudden change of Ca2+ concentration. In addition, the bioluminescence activities of Chinese hamster ovary cells expressing wild-type photoproteins also differed. The light signals of cells expressing mitrocomin, for example, slightly exceeded the background, suggesting that mitrocomin may be hardly used to detect intracellular Ca2+ without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y(2) receptor in Chinese hamster ovary cells by ATP, we suggest that wild-type aequorin and obelin from O. longissima are more suitable for calcium detection in cytoplasm, whereas clytin and obelin from O. geniculata can be used for calcium measurement in cell compartments with high Ca2+ concentration.
WOS
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva, Elena V., Vysotski, Eugene S.
Заглавие : Bioluminescent and biochemical properties of Cys-free Ca2+-regulated photoproteins obelin and aequorin
Колич.характеристики :9 с
Коллективы : Russian Academy of Sciences [03562016-0712, 0356-2015-0103]; RFBR [17-04-00764]
Место публикации : J. Photochem. Photobiol. B-Biol.: ELSEVIER SCIENCE SA, 2017. - Vol. 174. - С. 97-105. - ISSN 1011-1344, DOI 10.1016/j.jphotobio1.2017.07.021
Примечания : Cited References:54. - This work was supported by the state budget allocated to the fundamental research at the Russian Academy of Sciences (projects 03562016-0712 and 0356-2015-0103) and the RFBR grant 17-04-00764.
Предметные рубрики: SEQUENCE-ANALYSIS
APO-OBELIN
INTRINSIC FLUORESCENCE
COELENTERAZINE
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--photoprotein--coelenteramide--cysteine--serine
Аннотация: Bioluminescence of a variety of marine coelenterates is determined by Ca2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or p-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coil, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild -type aequorin. In contrast, Cys-free obelin retains only 10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a "fast" component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (Tm) were determined to be 53.3 0.2 and 44.6 0.4 C for aequorin and Cys-free aequorin, and 49.1 0.1 and 28.8 0.3 C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield.
WOS,
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva, Elena V., Bartsev, Sergey I., van Berkel, Willem J. H., Vysotski, Eugene S.
Заглавие : Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+-regulated Photoproteins of Different Organisms
Колич.характеристики :8 с
Коллективы : RFBR [14-04-31092]; Russian Academy of Sciences [01201351504, 01201351502]
Место публикации : Photochem. Photobiol.: WILEY, 2017. - Vol. 93, Is. 2. - С. 495-502. - ISSN 0031-8655, DOI 10.1111/php.12664. - ISSN 1751-1097(eISSN)
Примечания : Cited References:55. - This work was supported by RFBR grant 14-04-31092 and the state budget allocated to the fundamental research at the Russian Academy of Sciences (projects 01201351504 and 01201351502).
Предметные рубрики: GREEN-FLUORESCENT PROTEIN
AEQUORIN BIOLUMINESCENCE
SEQUENCE-ANALYSIS
Аннотация: Upon binding their metal ion cofactors, Ca2+-regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca2+-regulated photoproteinsaequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculatademonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca2+-regulated photoproteins.
WOS,
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Berestovskaya N.G., Shaloiko L.A., Gorokhovatsky A.Y., Bondar V.S., Vysotski E.S., Maximov J.E., von Doehren H..., Alakhov Y.B.
Заглавие : Cotranslational formation of active photoprotein obelin in a cell-free translation system: Direct ultrahigh sensitive measure of the translation course
Колич.характеристики :7 с
Место публикации : Anal. Biochem.: ACADEMIC PRESS INC, 1999. - Vol. 268, Is. 1. - P72-78. - ISSN 0003-2697, DOI 10.1006/abio.1998.3051
Примечания : Cited References: 22
Предметные рубрики: SEQUENCE-ANALYSIS
MESSENGER-RNA
CA-2+-ACTIVATED PHOTOPROTEIN
LIGHT-EMISSION
AEQUORIN
CDNA
CLONING
EXPRESSION
Аннотация: Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes. (C) 1999 Academic Press.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Berestovskaya N.G., Shaloiko L.A., Gorokhovatsky A.Y., Bondar V.S., Vysotski E.S., Maximov J.E., von Doehren H..., Alakhov Y.B.
Заглавие : Cotranslational formation of active photoprotein obelin in a cell-free translation system: Direct ultrahigh sensitive measure of the translation course
Колич.характеристики :7 с
Место публикации : Anal. Biochem.: ACADEMIC PRESS INC, 1999. - Vol. 268, Is. 1. - С. 72-78. - ISSN 0003-2697, DOI 10.1006/abio.1998.3051
Примечания : Cited References: 22
Предметные рубрики: SEQUENCE-ANALYSIS
MESSENGER-RNA
CA-2+-ACTIVATED PHOTOPROTEIN
LIGHT-EMISSION
AEQUORIN
CDNA
CLONING
EXPRESSION
Аннотация: Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes. (C) 1999 Academic Press.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova S.V., Burakova L.P., Frank L.A., Golz S..., Korostileva K.A., Vysotski E.S.
Заглавие : Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria: cDNA cloning, expression, and characterization of novel recombinant protein
Колич.характеристики :9 с
Коллективы :
Место публикации : Photochem. Photobiol. Sci.: ROYAL SOC CHEMISTRY, 2010. - Vol. 9, Is. 6. - С. 757-765. - ISSN 1474-905X, DOI 10.1039/c0pp00023j
Примечания : Cited References: 42. - We thank Dr John Lee (University of Georgia) for constructive suggestions. This work was supported by the Russian Foundation for Basic Research (Grants: 08-04-92209 and 09-04-12022), "Molecular and Cell Biology" program of RAS, and Bayer AG (Germany).
Предметные рубрики: ENERGY-TRANSFER
CA2+-REGULATED PHOTOPROTEINS
RENILLA BIOLUMINESCENCE
ANGSTROM RESOLUTION
SEQUENCE-ANALYSIS
CRYSTAL-STRUCTURE
EXCITED-STATE
AEQUORIN
PURIFICATION
OBELIN
Аннотация: The bioluminescent systems of many marine organisms are comprised of two proteins - the Ca2+-regulated photoprotein and green-fluorescent protein (GFP). This work reports the cloning of the full-size cDNA encoding GFP (cgreGFP) from jellyfish Clytia gregaria, its expression and properties of the recombinant protein. The overall degree of identity between the amino acid sequence of the novel cgreGFP and the sequence of GFP (avGFP) from Aequorea victoria is 42% (similarity - 64%) despite these GFPs originating from jellyfish that both belong to the same class, Hydrozoa. However although the degree of identity is low, three residues, Ser-Tyr-Gly, which form the chromophore are identical in both GFPs. The cgreGFP displayed two absorption peaks at 278 and 485 nm, and the fluorescence maximum at 500 nm. The fluorescence quantum yield was determined to be 0.86, the brightness to be 54 mM(-1) cm(-1). For the first time we have also demonstrated an efficient radiationless energy transfer in vitro between clytin and cgreGFP in solution at micromolar concentrations. The cgreGFP may be a useful intracellular fluorescent marker, as it was able to be expressed in mammalian cells.
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10.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova S.V., Burakova L.P., Golz S..., Malikova N.P., Frank L.A., Vysotski E.S.
Заглавие : The light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca2+-regulated photoprotein
Колич.характеристики :15 с
Место публикации : FEBS J.: WILEY-BLACKWELL, 2012. - Vol. 279, Is. 5. - С. 856-870. - ISSN 1742-464X, DOI 10.1111/j.1742-4658.2012.08476.x
Примечания : Cited References: 63. - The authors thank Natalia Chervyakova from Department of Zoology of Invertebrates of Moscow State University for the photo of the White Sea ctenophore Beroe abyssicola. This work was supported by RFBR grant 09-04-00172, by grant 64987.2010.4, Molecular and Cellular Biology program of RAS, and Bayer Pharma AG (Germany).
Предметные рубрики: CALCIUM-ACTIVATED PHOTOPROTEINS
C-TERMINAL PROLINE
SEQUENCE-ANALYSIS
MNEMIOPSIS-SP
COELENTERAZINE-BINDING
ANGSTROM RESOLUTION
RECOMBINANT OBELIN
CRYSTAL-STRUCTURES
EXCITED-STATE
CDNA CLONING
Ключевые слова (''Своб.индексиров.''): bioluminescence--calcium--coelenterazine--luciferase--mammalian expression
Аннотация: Light-sensitive Ca2+-regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca2+-regulated photoproteins, but a very low degree of sequence identity (2729%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca2+-discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (?max = 491 nm) and a change in pH over the range 6.09.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca2+-discharged protein (?ex = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca2+ concentrationeffect curve is a sigmoid with a slope on a loglog plot of similar to 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca2+-independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium.
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11.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva E.V., Markova S.V., Frank L.A., Visser AJWG, van Berkel WJH, Vysotski E.S.
Заглавие : Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin
Колич.характеристики :9 с
Место публикации : Photochem. Photobiol. Sci.: ROYAL SOC CHEMISTRY, 2013. - Vol. 12, Is. 6. - С. 1016-1024. - ISSN 1474-905X, DOI 10.1039/c3pp00002h
Примечания : Cited References: 46. - The work was supported by RFBR grant 12-04-00131, by the Programs of the Government of Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.0058), "Molecular and Cellular Biology" of RAS, President of Russian Federation "Leading science school" (grant 1044.2012.2). E.V.E. was supported by Wageningen University Sandwich PhD-Fellowship Program.
Предметные рубрики: CA2+-REGULATED PHOTOPROTEINS
CA2+-BINDING PHOTOPROTEIN
SEQUENCE-ANALYSIS
CRYSTAL-STRUCTURE
VIOLET BIOLUMINESCENCE
ANGSTROM RESOLUTION
MNEMIOPSIS-LEIDYI
LIGHT-EMISSION
W92F OBELIN
CLONING
Аннотация: Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor-acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His-Trp-Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His-Trp-Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different.
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12.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Deng L..., Vysotski E.S., Markova S.V., Liu Z.J., Lee J..., Rose J..., Wang B.C.
Заглавие : All three Ca2+-binding loops of photoproteins bind calcium ions: The crystal structures of calcium-loaded apo-aequorin and apo-obelin
Колич.характеристики :13 с
Место публикации : Protein Sci.: COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2005. - Vol. 14, Is. 3. - С. 663-675. - ISSN 0961-8368, DOI 10.1110/ps.041142905
Примечания : Cited References: 46
Предметные рубрики: RAY CRYSTALLOGRAPHIC ANALYSIS
ANGSTROM RESOLUTION
SEQUENCE-ANALYSIS
CA2+-REGULATED PHOTOPROTEINS
CA2+-DISCHARGED PHOTOPROTEIN
LUMINESCENT PROTEIN
MODULATED PROTEINS
ELECTRON-DENSITY
CLONING
CDNA
Ключевые слова (''Своб.индексиров.''): bioluminescence--ef-hand--fluorescent protein--proton relay--calcium-binding loops--aequorin--obelin--diffraction
Аннотация: The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7 Angstrom and 2.2 Angstrom. respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hydroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.
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13.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Illarionov B.A., Frank L.A., Illarionova V.A., Bondar V.S., Vysotski E.S., Blinks J.R.
Заглавие : Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator
Колич.характеристики :27 с
Место публикации : Methods Enzymol.: ACADEMIC PRESS INC, 2000. - Vol. 305. - С. 223-249. - ISSN 0076-6879
Примечания : Cited References: 58
Предметные рубрики: PHOTOPROTEIN OBELIN
MESSENGER-RNA
CA-2+-ACTIVATED PHOTOPROTEIN
DIRECTED MUTAGENESIS
SEQUENCE-ANALYSIS
HYDROID OBELIA
AEQUORIN
PROTEIN
BIOLUMINESCENCE
LUMINESCENCE
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14.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Vysotski E.S., Lee J...
Заглавие : Ca2+-regulated photoproteins: Structural insight into the bioluminescence mechanism
Колич.характеристики :11 с
Место публикации : Accounts Chem. Res.: AMER CHEMICAL SOC, 2004. - Vol. 37, Is. 6. - С. 405-415. - ISSN 0001-4842, DOI 10.1021/ar0400037
Примечания : Cited References: 44
Предметные рубрики: AEQUORIN BIOLUMINESCENCE
SEQUENCE-ANALYSIS
CALCIUM-BINDING
CA2+-BINDING PHOTOPROTEIN
VIOLET BIOLUMINESCENCE
ANGSTROM RESOLUTION
FUNCTIONAL PART
EXCITED-STATES
W92F OBELIN
COELENTERAZINE
Аннотация: The bioluminescent jellyfish has contributed two famous proteins to modern science: green fluorescent protein or GFP, which finds wide use as a probe in cell biology studies, and aequorin, which has been used for intracellular calcium measurement for more than 30 years. More recently, obelin, a protein from the bioluminescent hydroid and also in the family of what are called "Ca2+-regulated photoproteins", has been shown to have very attractive properties both in general applications and for basic structural biology investigations. This review will survey the new information into their molecular mechanism of bioluminescence action.
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15.