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PHYSICOCHEMICAL PROPERTIES OF A PHOTOPROTEIN FROM THE HYDROID POLYP OBELIA-LONGISSIMA [Text] / V. S. BONDAR, K. P. TROFIMOV, E. S. VYSOTSKII // Biochem.-Moscow. - 1992. - Vol. 57, Is. 10. - P1020-1027. - Cited References: 36 . - 8. - ISSN 0006-2979

РУБ Biochemistry & Molecular Biology
Рубрики:
CALCIUM-ACTIVATED PHOTOPROTEINS
   CTENOPHORES MNEMIOPSIS SP
   BEROE-OVATA
   AEQUORIN
   CA-2+
   INDICATORS
   PROTEIN
   BINDING
   PURIFICATION
   EXTRACTION
Кл.слова (ненормированные):
BIOLUMINESCENCE -- CA2+-ACTIVATED PHOTOPROTEIN -- OBELIN -- CHROMATOGRAPHY -- CALCIUM
Аннотация: The photoprotein obelin was isolated and purified to homogeneity (as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis) from hydroids of Obelia longissima by gel filtration on Sephadex G-75 fine, ion exchange chromatography on Polysil CA-300 (10 mum), hydrophobic chromatography on Phenyl-Sepharose CL-4B, gel filtration on Sephacryl S-200 superfine, ion exchange chromatography on a Mono Q column at pH 7.0, chromatofocusing on a Mono P column (pH gradient 6.0-4.0), and ion exchange chromatography on a Mono Q column at pH 5.5, 8.8, and 7.0. The molecular weight of the native protein was 30 kD, and that measured in the presence of SDS was 19.8 kD. The specific activity of obelin is 4.9.10(15) quanta/mg protein, pseudo-first-order constant of bioluminescence decay 4 sec-1, and quantum yield 0.16 The range of measurable Ca2+ concentrations is 10(-7) to 10(-5) M. The luminescence spectrum of obelin peaks at 469 nm, and the fluorescence emission maximum of the discharged protein is at 455 nm. The optimum pH for luminescence is between 9.0 and 10.5. The molecular ionization constants are pK1 6.8 and pK2 12.2, and the ionization constants for the active site are pK1 9.1 and pK2 10.2
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
BONDAR, V.S.; TROFIMOV, K.P.; VYSOTSKII, E.S.

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SEQUENCE OF THE CDNA-ENCODING THE CA2+-ACTIVATED PHOTOPROTEIN OBELIN FROM THE HYDROID POLYP OBELIA-LONGISSIMA [Text] / B. A. ILLARIONOV [et al.] // Gene. - 1995. - Vol. 153, Is. 2. - P273-274, DOI 10.1016/0378-1119(94)00797-V. - Cited References: 6 . - 2. - ISSN 0378-1119

РУБ Genetics & Heredity
Рубрики:
CA-2+-ACTIVATED PHOTOPROTEIN
AEQUORIN
CLONING
Кл.слова (ненормированные):
BIOLUMINESCENCE -- CALCIUM -- GENE -- PLASMID -- MARINE COELENTERATES
Аннотация: A cDNA clone encoding the Ca2+-activated photoprotein, obelin (Obl), from Obelia longissima was sequenced. The nucleotide (nt) sequence contained two long overlapping open reading frames (ORFs), one of which encoded apoobelin (apoObl). The deduced amino acid (aa) sequence of apoObl revealed that this 195-aa protein has three EF-hand structures that are characteristic for Ca2+-binding domains. Strong aa homology was shown among apoObl, apoaequorin and apoclytin. The second ORF present in the obl cDNA consists of 139 codons and encodes a very basic protein with a calculated pI of 10.56 and a molecular mass of 16153 Da.
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
ILLARIONOV, B.A.; BONDAR, V.S.; ILLARIONOVA, V.A.; VYSOTSKI, E.S.

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MN2+-ACTIVATED LUMINESCENCE OF THE PHOTOPROTEIN OBELIN [Text] / E. S. VYSOTSKI [et al.] // Arch. Biochem. Biophys. - 1995. - Vol. 316, Is. 1. - P92-99, DOI 10.1006/abbi.1995.1014. - Cited References: 38 . - 8. - ISSN 0003-9861

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
CALCIUM-ACTIVATED PHOTOPROTEINS
   CTENOPHORES MNEMIOPSIS SP
   CA-2+-ACTIVATED PHOTOPROTEIN
   MESSENGER-RNA
   BEROE-OVATA
   AEQUORIN
   PURIFICATION
   PROTEIN
   CDNA
   EXTRACTION
Аннотация: The light emission of obelin may be initiated by Mn2+ under alkaline conditions. The luminescence takes place in a pH range from 7 to 12 with a sharp optimum at 11.75. The first-order rate constant for Mn2+-activated luminescence decay is more than 9 s(-1), while that for Ca2+-activated luminescence decay is only 6.9 s(-1). The Mn2+ concentration-effect curve for obelin determined with simple dilutions of manganese salt is a sigmoid curve, The slope of the curve is moderately dependent on the pH and was not more than 1 within the pH range tested. The maximal light emission, which is initiated by 3.6 X 10(-5) M Mn2+ at pH 11.75 was about 10% of the maximal Ca2+-activated luminescence. Mg2+ ions inhibit the Mn2+-activated luminescence of obelin. The addition of OH. and O-2(-) scavengers did not influence the Mn2+-activated luminescence, but when singlet oxygen quenchers were added, the Mn2+-dependent light emission was inhibited. This suggests that the O-1(2) might be formed and itself be responsible for chromophore oxidation attended with light emission. NEM and Na2S2O4 inhibit the Mn2+-initiated light emission of obelin completely, showing that endogenous hydroperoxide and SH-group(s) of the photoprotein are essential for both Ca2+-activated and Mn2+-activated light emission of obelin. (C) 1995 Academic Press, Inc.
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
VYSOTSKI, E.S.; TROFIMOV, C.P.; BONDAR, V.S.; FRANK, L.A.; MARKOVA, S.V.; ILLARIONOV, B.A.

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Use of proZZ-obelin fusion protein in bioluminescent immunoassay [Text] / L. A. Frank, V. A. Illarionova, E. S. Vysotski // Biochem. Biophys. Res. Commun. - 1996. - Vol. 219, Is. 2. - P475-479, DOI 10.1006/bbrc.1996.0258. - Cited References: 21 . - 5. - ISSN 0006-291X

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
ESCHERICHIA-COLI
   EXPRESSION
   AEQUORIN
   PURIFICATION
   SYSTEM
Аннотация: Obelin is a photoprotein that emits light by Ca2+-binding. To develop a bioluminescent immunoassay based on the light emission property of obelin, we have expressed the apoobelin fusion protein with ZZ-domain of S. aureus protein A in E. coil by recombinant DNA techniques. The pro2Z-obelin expressed was purified by one-step affinity chromatography on IgG-Agarose. The purified proZZ-obelin has both the luminescent activity of obelin and the IgG-binding ability of ZZ-domain. The specific activity of fusion protein was 8.5 x 10(15) photons per mg of protein. (C) 1996 Academic Press, Inc.
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Illarionova, V.A.; Vysotski, E.S.

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ISOLATION AND EXPRESSION OF CDNA CODING FOR PHOTOPROTEIN OBELIN FROM HYDROID OBELIA-LONGISSIMA [Текст] / B. A. ILLARIONOV [и др.] // Dokl. Akad. Nauk. - 1992. - Vol. 326, Is. 5. - С. 911-913. - Cited References: 12 . - 3. - ISSN 0869-5652

РУБ Multidisciplinary Sciences
Рубрики:
AEQUORIN
   PROTEIN
   PHIALIDIN
   CLONING
   CA-2+
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
ILLARIONOV, B.A.; MARKOVA, S.V.; BONDAR, V.S.; VYSOTSKY, E.S.; GITELSON, J.I.

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Oxygen Activation of Apo-obelin-Coelenterazine Complex / E. V. Eremeeva [et al.] // ChemBioChem. - 2013. - Vol. 14, Is. 6. - P739-745, DOI 10.1002/cbic.201300002. - Cited References: 46. - The work was supported by grants from the RFBR 12-04-91153, and NSFC 31270795 and 31021062, by the Russian Federation Government Program "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.0058), "Molecular and Cellular Biology" of RAS, President of the Russian Federation "Leading Science School" (grant 1044.2012.2). E.V.E. was supported by a Wageningen University Sandwich PhD Fellowship Program. . - ISSN 1439-4227

РУБ Biochemistry & Molecular Biology + Chemistry, Medicinal
Рубрики:
GREEN-FLUORESCENT PROTEIN
   JELLYFISH CLYTIA-GREGARIA
   CRYSTAL-STRUCTURE
   CA2+-DISCHARGED PHOTOPROTEIN
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   MOLECULAR-OXYGEN
   AEQUORIN
   CALCIUM
   BIOLUMINESCENCE
Кл.слова (ненормированные):
aequorin -- coelenterazine -- luminescence -- photoprotein -- protein folding
Аннотация: Ca2+-regulated photoproteins use a noncovalently bound 2-hydroperoxycoelenterazine ligand to emit light in response to Ca2+ binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo-obelincoelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7-protonated and C2() anionic forms, and that oxygen shifts the equilibrium in favor of the C2() anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2-hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca2+-triggering of the bioluminescence reaction.

Держатели документа:
[Eremeeva, Elena V.
Natashin, Pavel V.
Liu, Zhi-Jie] Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
[Eremeeva, Elena V.
Natashin, Pavel V.
Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Natashin, Pavel V.
Vysotski, Eugene S.] Siberian Fed Univ, Lab Bioluminescence Biotechnol, Inst Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia
[Eremeeva, Elena V.
Natashin, Pavel V.
Vysotski, Eugene S.] Siberian Fed Univ, Chair Biophys, Inst Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia
[Eremeeva, Elena V.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
[Song, Lei
Zhou, Yuguang] Chinese Acad Sci, China Gen Microbiol Culture Collect Ctr, Inst Microbiol, Beijing 100101, Peoples R China
[Liu, Zhi-Jie] Kunming Med Univ, Inst Mol & Clin Med, Kunming 650500, Peoples R China
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Natashin, P.V.; Song, L...; Zhou, Y.G.; van Berkel, WJH; Liu, Z.J.; Vysotski, E.S.

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Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin / E. V. Eremeeva [et al.] // Photochem. Photobiol. Sci. - 2013. - Vol. 12, Is. 6. - P1016-1024, DOI 10.1039/c3pp00002h. - 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. . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
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.

Держатели документа:
[Eremeeva, Elena V.
Markova, Svetlana V.
Frank, Ludmila A.
Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Visser, Antonie J. W. G.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
[Eremeeva, Elena V.
Markova, Svetlana V.
Frank, Ludmila A.
Vysotski, Eugene S.] Siberian Fed Univ, Inst Fundamental Biol & Biotechnol, Lab Bioluminescence Biotechnol, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Frank, L.A.; Visser, AJWG; van Berkel, WJH; Vysotski, E.S.

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Ligand binding and conformational states of the photoprotein obelin / E. V. Eremeeva [et al.] // FEBS Lett. - 2012. - Vol. 586, Is. 23. - P4173-4179, DOI 10.1016/j.febslet.2012.10.015. - Cited References: 24. - The work was supported by RFBR grant 12-04-00131, by the Program of the Government of Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.058), by the Program "Molecular and Cellular Biology" of RAS. The Wageningen University Sandwich PhD-Fellowship Program supported E.V.E. . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
RECOMBINANT OBELIN
   CRYSTAL-STRUCTURE
   LIGHT-EMISSION
   APO-AEQUORIN
   BIOLUMINESCENCE
   COELENTERAZINE
   LUMINESCENCE
   STABILITY
   ANGSTROM
   PROTEINS
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Photoprotein -- Thermostability
Аннотация: Many proteins require a non-covalently bound ligand to be functional. How ligand binding affects protein conformation is often unknown. Here we address thermal unfolding of the free and ligand-bound forms of photoprotein obelin. Fluorescence and far-UV circular dichroism ( CD) data show that the various ligand-dependent conformational states of obelin differ significantly in stability against thermal unfolding. Binding of coelenterazine and calcium considerably stabilizes obelin. In solution, all obelin structures are similar, except for apo-obelin without calcium. This latter protein is an ensemble of conformational states, the populations of which alter upon increasing temperature. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Держатели документа:
[Eremeeva, Elena V.
Westphal, Adrie H.
van Mierlo, Carlo P. M.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
[Eremeeva, Elena V.
Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Vysotski, Eugene S.] Siberian Fed Univ, Lab Bioluminescence Biotechnol, Inst Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Vysotski, E.S.; Westphal, A.H.; van Mierlo, CPM; van Berkel, WJH

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Sensitivity of Ca2+-regulated photoprotein bioluminescence to magnesium ions is determined by EF-hand motif III / L. P. Burakova, N. P. Malikova, E. S. Vysotski // Luminescence. - 2012. - Vol. 27, Is. 2. - P102-103. - Cited References: 3 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology
Рубрики:
AEQUORIN

Держатели документа:
[Burakova, L. P.
Malikova, N. P.
Vysotski, E. S.] Inst Biophys SB RAS, Photobiol Lab, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Burakova, L.P.; Malikova, N.P.; Vysotski, E.S.

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10.

The light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca2+-regulated photoprotein / S. V. Markova [et al.] // FEBS J. - 2012. - Vol. 279, Is. 5. - P856-870, DOI 10.1111/j.1742-4658.2012.08476.x. - 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). . - ISSN 1742-464X

РУБ Biochemistry & Molecular Biology
Рубрики:
CALCIUM-ACTIVATED PHOTOPROTEINS
   C-TERMINAL PROLINE
   SEQUENCE-ANALYSIS
   MNEMIOPSIS-SP
   COELENTERAZINE-BINDING
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   CRYSTAL-STRUCTURES
   EXCITED-STATE
   CDNA CLONING
Кл.слова (ненормированные):
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.

Держатели документа:
[Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Markova, Svetlana V.
Burakova, Ludmila P.
Malikova, Natalia P.
Frank, Ludmila A.
Vysotski, Eugene S.] Siberian Fed Univ, Dept Biophys, Krasnoyarsk, Russia
[Golz, Stefan] Bayer Pharma AG, Global Drug Discovery, Wuppertal, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Burakova, L.P.; Golz, S...; Malikova, N.P.; Frank, L.A.; Vysotski, E.S.

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11.

NMR-derived Topology of a GFP-photoprotein Energy Transfer Complex [Text] / M. S. Titushin [et al.] // J. Biol. Chem. - 2010. - Vol. 285, Is. 52. - P40891-40900, DOI 10.1074/jbc.M110.133843. - Cited References: 54. - This work was supported by the National Natural Science Foundation of China, Ministry of Science and Technology of China, CAS Research Grant, CAS Fellowship for Young International Scientists Grant, Russian Foundation for Basic Research (08-09-92209 RFBR-China joint grant), SB RAS Grant 2, "Molecular and Cell Biology" program of RAS, Bayer AG (Germany), and by the University of Georgia Research Foundation and the Georgia Research Alliance. . - ISSN 0021-9258

РУБ Biochemistry & Molecular Biology
Рубрики:
GREEN-FLUORESCENT PROTEIN
   STRUCTURAL DETERMINANTS
   RENILLA BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   CRYSTAL-STRUCTURE
   ELECTRON-DENSITY
   SOFTWARE
   PROGRAM
   BINDING
   SYSTEM
Аннотация: Forster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca2+ regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously N-15, C-13, H-2-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain H-1-N-15 HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (K-D = 0.9 mM). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.

Держатели документа:
[Wang, Jinfeng] Chinese Acad Sci, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China
[Titushin, Maxim S.
Stepanyuk, Galina A.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Lab Photobiol, Krasnoyarsk 660036, Russia
[Golz, Stefan] Bayer Schering Pharma AG, BSP GDD GTR TD GT, D-42096 Wuppertal, Germany
[Stepanyuk, Galina A.
Wang, Bi-Cheng
Lee, John] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Titushin, M.S.; Feng, Y.G.; Stepanyuk, G.A.; Li, Y...; Markova, S.V.; Golz, S...; Wang, B.C.; Lee, J...; Wang, J.F.; Vysotski, E.S.; Liu, Z.J.

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12.

Quantum chemical study of mechanism of active photoprotein generation [Text] / F. N. Tomilin [et al.] // Luminescence. - 2010. - Vol. 25, Is. 2. - P210-211. - Cited References: 4 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology
Рубрики:
AEQUORIN

Держатели документа:
[Eremeeva, E. V.
Vysotski, E. S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Tomilin, F. N.
Ovchinnikov, S. G.] Siberian Fed Univ, Krasnoyarsk 660036, Russia
[Tomilin, F. N.
Antipina, L. U.
Ovchinnikov, S. G.] Russian Acad Sci, Inst Phys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН
ИФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Tomilin, F.N.; Antipina, L.U.; Eremeeva, E.V.; Ovchinnikov, S.G.; Vysotski, E.S.

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13.

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria: cDNA cloning, expression, and characterization of novel recombinant protein [Text] / S. V. Markova [et al.] // Photochem. Photobiol. Sci. - 2010. - Vol. 9, Is. 6. - P757-765, DOI 10.1039/c0pp00023j. - 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). . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
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.

Держатели документа:
[Markova, Svetlana V.
Burakova, Ludmila P.
Frank, Ludmila A.
Korostileva, Kseniya A.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Photobiol Lab, Krasnoyarsk 660036, Russia
[Markova, Svetlana V.
Frank, Ludmila A.
Korostileva, Kseniya A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
[Golz, Stefan] Bayer Schering Pharma AG, BSP GDD GTR TD GT, D-42096 Wuppertal, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Burakova, L.P.; Frank, L.A.; Golz, S...; Korostileva, K.A.; Vysotski, E.S.

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14.

Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay [Text] / L. A. Frank [et al.] // Anal. Bioanal. Chem. - 2008. - Vol. 391, Is. 8. - P2891-2896, DOI 10.1007/s00216-008-2223-5. - Cited References: 22 . - ISSN 1618-2642

РУБ Biochemical Research Methods + Chemistry, Analytical
Рубрики:
ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   CRYSTAL-STRUCTURE
   BIOLUMINESCENCE
   AEQUORIN
   IMMUNOASSAY
   EXPRESSION
   CDNA
   PURIFICATION
   CLONING
Кл.слова (ненормированные):
Ca(2+)-regulated photoprotein -- bioluminescence -- dual-color assay
Аннотация: Two kinds of Ca(2+)-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (lambda(max)=390 nm) and the mutant Y139F emits greenish light (lambda (max)=498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones-follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca(2+) solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.

Держатели документа:
[Frank, Ludmila A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
[Frank, Ludmila A.
Borisova, Vasilisa V.
Markova, Svetlana V.
Malikova, Natalia P.
Stepanyuk, Galina A.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Borisova, V.V.; Markova, S.V.; Malikova, N.P.; Stepanyuk, G.A.; Vysotski, E.S.

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15.

Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 angstrom: Why it is not a calcium-regulated photoprotein [Text] / G. A. Stepanyuk [et al.] // Photochem. Photobiol. Sci. - 2008. - Vol. 7, Is. 4. - P442-447, DOI 10.1039/b716535h. - Cited References: 49 . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
HYDROID OBELIA-GENICULATA
   AMINO-ACID-SEQUENCE
   CA2+-REGULATED PHOTOPROTEINS
   RENIFORMIS LUCIFERASE
   ENERGY-TRANSFER
   CDNA CLONING
   BIOLUMINESCENCE
   AEQUORIN
   PURIFICATION
   EXPRESSION
Аннотация: Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O-2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three "EF-hand" Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 angstrom, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity are proposed to account for the different properties of the two classes of proteins.

Держатели документа:
[Liu, Zhi-Jie] Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
[Stepanyuk, Galina A.
Lee, John
Vysotski, Eugene S.
Wang, Bi-Cheng] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
[Stepanyuk, Galina A.
Markova, Svetlana S.
Frank, Ludmila A.
Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Photobiol Lab, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Liu, Z.J.; Markova, S.S.; Frank, L.A.; Lee, J...; Vysotski, E.S.; Wang, B.C.

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16.

The kinetics of coelenterazine binding with apo-obelin and apo-aequorin [Text] / E. V. Eremeeva [et al.] // Luminescence. - 2008. - Vol. 23, Is. 2. - P66-67. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Eremeeva, E. V.
Markova, S. V.
Frank, L. A.
Vysotski, E. S.] SB RAS, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Lee, J.
Vysotski, E. S.] Univ Georgia, Dept Mol Biol & Biochem, Athens, GA 30602 USA
[Eremeeva, E. V.
van Berkel, W. J. H.
Visser, A. J. W. G.] Univ Wageningen & Res Ctr, Biochem Lab, NL-6703 HA Wageningen, Netherlands
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Frank, L.A.; Lee, J...; van Berkel, WJH; Visser, AJWG; Vysotski, E.S.

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17.

All three Ca2+-binding loops of photoproteins bind calcium ions: The crystal structures of calcium-loaded apo-aequorin and apo-obelin [Text] / L. . Deng [et al.] // Protein Sci. - 2005. - Vol. 14, Is. 3. - P663-675, DOI 10.1110/ps.041142905. - Cited References: 46 . - ISSN 0961-8368

РУБ Biochemistry & Molecular Biology
Рубрики:
RAY CRYSTALLOGRAPHIC ANALYSIS
   ANGSTROM RESOLUTION
   SEQUENCE-ANALYSIS
   CA2+-REGULATED PHOTOPROTEINS
   CA2+-DISCHARGED PHOTOPROTEIN
   LUMINESCENT PROTEIN
   MODULATED PROTEINS
   ELECTRON-DENSITY
   CLONING
   CDNA
Кл.слова (ненормированные):
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.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Univ Georgia, Dept Chem, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Deng, L...; Vysotski, E.S.; Markova, S.V.; Liu, Z.J.; Lee, J...; Rose, J...; Wang, B.C.

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18.

Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein [Text] / G. A. Stepanyuk [et al.] // FEBS Lett. - 2005. - Vol. 579, Is. 5. - P1008-1014, DOI 10.1016/j.febslet.2005.01.004. - Cited References: 49 . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
FIREFLY LUCIFERASE
   SEQUENCE-ANALYSIS
   CA2+-REGULATED PHOTOPROTEINS
   CA2+-DISCHARGED PHOTOPROTEIN
   VIOLET BIOLUMINESCENCE
   INTRACELLULAR CALCIUM
   ENDOPLASMIC-RETICULUM
   ANGSTROM RESOLUTION
   CRYSTAL-STRUCTURE
   APOAEQUORIN CDNA
Кл.слова (ненормированные):
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.

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
Bayer AG, Pharma Res Mol Screening Technol, D-42096 Wuppertal, Germany
Univ Georgia, Dept Mol Biol & Biochem, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Golz, S...; Markova, S.V.; Frank, L.A.; Lee, J...; Vysotski, E.S.

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19.

Crystal structure of a Ca2+-discharged photoprotein - Implications for mechanisms of the calcium trigger and bioluminescence [Text] / L. . Deng [et al.] // J. Biol. Chem. - 2004. - Vol. 279, Is. 32. - P33647-33652, DOI 10.1074/jbc.M402427200. - Cited References: 31 . - ISSN 0021-9258

РУБ Biochemistry & Molecular Biology
Рубрики:
VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   ELECTRON-DENSITY
   W92F OBELIN
   AEQUORIN
   PROTEINS
   LIGHT
   SEQUENCE
   BINDING
   COELENTERAZINE
Аннотация: Ca2+-regulated photoproteins are members of the EF-hand calcium-binding protein family. The addition of Ca2+ produces a blue bioluminescence by triggering a decarboxylation reaction of protein-bound hydroperoxycoelenterazine to form the product, coelenteramide, in an excited state. Based on the spatial structures of aequorin and several obelins, we have postulated mechanisms for the Ca2+ trigger and for generation of the different excited states that are the origin of the different colors of bioluminescence. Here we report the crystal structure of the Ca2+-discharged photoprotein obelin at 1.96-Angstrom resolution. The results lend support to the proposed mechanisms and provide new structural insight into details of these processes. Global conformational changes caused by Ca2+ association are typical of the class of calcium signal modulators within the EF-hand protein superfamily. Accommodation of the Ca2+ ions into the loops of the EF-hands is seen to propagate into the active site of the protein now occupied by the coelenteramide where there is a significant repositioning and flipping of the His-175 imidazole ring as crucially required in the trigger hypothesis. Also the H-bonding between His-22 and the coelenterazine found in the active photoprotein is preserved at the equivalent position of coelenteramide, confirming the proposed rapid excited state proton transfer that would lead to the excited state of the phenolate ion pair, which is responsible for the blue emission of bioluminescence.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Univ Georgia, Dept Chem, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Deng, L...; Markova, S.V.; Vysotski, E.S.; Liu, Z.J.; Lee, J...; Rose, J...; Wang, B.C.

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20.

Conjugates of the Ca2+-regulated photoprotein obelin with immunoglobulins: Synthesis and use as labels in bioluminescent immunoassay [Text] / L. A. Frank, A. I. Petunin, E. S. Vysotski // Russ. J. Bioorg. Chem. - 2004. - Vol. 30, Is. 4. - P327-331, DOI 10.1023/B:RUBI.0000037257.80835.7a. - Cited References: 16 . - ISSN 1068-1620

РУБ Biochemistry & Molecular Biology + Chemistry, Organic
Рубрики:
ESCHERICHIA-COLI
   PURIFICATION
   AEQUORIN
   PROTEIN
   CDNA
Кл.слова (ненормированные):
bioluminescent immunoassay -- obelin -- thyroid stimulating hormone
Аннотация: An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 muIU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
DIAS Ltd, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Petunin, A.I.; Vysotski, E.S.

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