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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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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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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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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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10.

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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11.

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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12.

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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13.

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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14.

Preparation and X-ray crystallographic analysis of the Ca2+-discharged photoprotein obelin [Text] / L. . Deng [et al.] // Acta Crystallogr. Sect. D-Biol. Crystallogr. - 2004. - Vol. 60. - P512-514, DOI 10.1107/S090744490302852X. - Cited References: 18 . - ISSN 0907-4449

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Biophysics + Crystallography
Рубрики:
VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   W92F OBELIN
   AEQUORIN
   SEQUENCE
   PROTEIN
   CLONING
   CDNA
Аннотация: Ca2+-regulated photoproteins belong to the EF-hand Ca2+-binding protein family. The addition of calcium ions initiates bright blue bioluminescence of the photoproteins, a result of the oxidative breakdown of coelenterazine peroxide to coelenteramide. Crystals of the Ca2+-discharged W92F mutant of obelin from Obelia longissima have been grown, representing the first crystallization of a photoprotein after the Ca2+-triggered bioluminescence. A green fluorescence observed from the crystals clearly demonstrates that coelenteramide, the bioluminescence product of coelenterazine peroxide, is bound within the protein. The diffraction pattern exhibits tetragonal Laue symmetry. Systematic absences indicate that the space group is either P4(3)2(1)2 or P4(1)2(1)2. The unit-cell parameters are a=b=53.4, c=144.0 Angstrom. The crystals diffract to 1.9 Angstrom resolution.

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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15.

Bioluminescent immunoassay of thyrotropin and thyroxine using obelin as a label [Text] / L. A. Frank, A. I. Petunin, E. S. Vysotski // Anal. Biochem. - 2004. - Vol. 325, Is. 2. - P240-246, DOI 10.1016/j.ab.2003.11.003. - Cited References: 16 . - ISSN 0003-2697

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Chemistry, Analytical
Рубрики:
AEQUORIN
   PURIFICATION
   PROTEIN
   CDNA
Кл.слова (ненормированные):
obelin -- human thyrotropin -- thyroxine -- immunoassay -- bioluminescence
Аннотация: Solid-phase bioluminescent immunoassay of thyroid hormones, human thyrotropin (hTSH) and two forms of thyroxine (T4), whose determinations are vitally important for diagnostics of thyroid diseases and the efficiency of treatment, is described. The recombinant obelin, a Ca2+-regulated photoprotein originally derived from the luminous marine hydroid Obelia longissima, is employed as a bioluminescent label. To produce obelin conjugates with anti-hTSH, anti-T4 immunoglobulins (IgG), and T4, additional SH groups are introduced into the obelin molecule using Traut's reagent (2-iminothiolane) and then obelin possessing extra SH groups is conjugated with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate-activated IgGs or T4. The total yield of obelin conjugates determined by luminescent activity is 60-65% after all chemical and purification procedures. The obtained conjugates are stable to lyophilization and in solution for at least 9 months at 4 degreesC, with loss of activity not exceeding 10%. The application of obelin conjugates for determination of the hTSH, total T4, and free T4 in standard, control, and patient sera displays high sensitivity and reproducibility of results. The results of bioluminescent immunoassays are closely comparable to those obtained by the radioimmunoassay method (R = 0.95-0.99). (C) 2003 Elsevier Inc. All rights reserved.

Держатели документа:
Russian Acad Sci, Inst Biophys, Siberian Branch, 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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16.

Spectral tuning of obelin bioluminescence by mutations of Trp92 [Text] / N. P. Malikova [et al.] // FEBS Lett. - 2003. - Vol. 554, Is. 01.02.2013. - P184-188, DOI 10.1016/S0014-5793(03)01166-9. - Cited References: 13 . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
VIOLET BIOLUMINESCENCE
   W92F OBELIN
   AEQUORIN
   PURIFICATION
   EXPRESSION
   EMISSION
   CLONING
Кл.слова (ненормированные):
photoprotein -- aequorin -- calcium -- fluorescence
Аннотация: The Ca2+-regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence (lambda(max)=390 nm) and monomodal fluorescence (lambda(max)=425 nm) of the product. Results are interpreted by an excited state proton transfer mechanism involving the substituent side group and His22 in the binding cavity. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
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:
Malikova, N.P.; Stepanyuk, G.A.; Frank, L.A.; Markova, S.V.; Vysotski, E.S.; Lee, J...

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17.

Obelin from the bioluminescent marine hydroid Obelia geniculata: Cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins [Text] / S. V. Markova [et al.] // Biochemistry. - 2002. - Vol. 41, Is. 7. - P2227-2236, DOI 10.1021/bi0117910. - Cited References: 54 . - ISSN 0006-2960

РУБ Biochemistry & Molecular Biology
Рубрики:
AMINO-ACID-SEQUENCE
   CALCIUM-ACTIVATED PHOTOPROTEINS
   CTENOPHORES MNEMIOPSIS SP
   CA-2+-ACTIVATED PHOTOPROTEIN
   LUMINESCENT PROTEIN
   BINDING-PROTEIN
   BEROE-OVATA
   AEQUORIN
   CDNA
   PURIFICATION
Аннотация: A cDNA encoding the Ca2+-regulated photoprotein of the bioluminescent marine hydroid Obelia geniculata was cloned and sequenced. The cDNA is a 774 bp fragment containing two overlapping open reading frames, one of which contained 585 bp encoding a 195 amino acid polypeptide which obviously has the primary structure of the apoprotein of a calcium-regulated photoprotein. Many of the residues are identical to those in other Ca2+-regulated photoproteins: 86% compared with that from Obelia longissima, 76% with that from Clytia (Phialidium), 64% with that from Aequorea, and 64% with that from Mitrocoma (Halistaura). The obelin from O. geniculata was overexpressed in Escherichia coli, refolded from inclusion bodies, and purified. The yield of highly purified recombinant protein was 55-80 mg/L of LB medium. O. geniculata obelin has absorption maxima at 280 and 460 nm and a shoulder at approximately 310 nm. The calcium-discharged protein loses visible absorption but exhibits a new absorption maximum at 343 nm. The bioluminescence of the obelin from O. geniculata is blue (lambda(max) = 495 nm). In contrast, the fluorescence of the calcium-discharged protein is yellow-green (lambda(max) = 520 nm; excitation at 340 nm). This is in sharp contrast to aequorin in which the bioluminescence and fluorescence emission spectra of the calcium-discharged protein are almost identical (lambda(max) = 465 nm). The Ca2+ concentration-effect curve for O. geniculata obelin is similar to those of many other photoproteins: at [Ca2+] below approximately 10(-8) M, calcium-independent luminescence is observed, and at [Ca2+] approximately 10(-3) M, the luminescence reaches a maximum. Between these extremes, the curve spans a vertical range of almost 8 log units with a maximum slope on a log-log plot of about 2.5. In the absence of Mg2+ the rate constant for the rise of bioluminescence determined by the stopped-flow technique is about 450 s(-1). The effects of Mg2+ on the kinetics of bioluminescence are complicated, but at all concentrations studied they are relatively small compared to the corresponding effects on aequorin luminescence. At least with respect to speed and sensitivity to Mg2+, the obelins from both O. longissima and O. geniculata would appear to be more suitable than aequorin for use as intracellular Ca2+ indicators.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
Univ Washington, Friday Harbor Labs, Friday Harbor, WA 98250 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Vysotski, E.S.; Blinks, J.R.; Burakova, L.P.; Wang, B.C.; Lee, J...

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18.

Structural basis for the emission of violet bioluminescence from a W92F obelin mutant [Text] / L. . Deng [et al.] // FEBS Lett. - 2001. - Vol. 506, Is. 3. - P281-285, DOI 10.1016/S0014-5793(01)02937-4. - Cited References: 15 . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
AEQUORIN
Кл.слова (ненормированные):
calcium-regulated photoprotein -- X-ray crystallography -- fluorescence -- coelenterazine -- aequorin
Аннотация: Mutation of the Trp92 that is known to lie within the active site of the photoprotein obelin from Obelia longissima, results in a shift of the bioluminescence color from blue (lambda (max) = 485 nm) to violet. The corrected spectrum shows a new band with lambda (max) = 410 nm now contributing equally to the one at longer wavelength. The crystal structure of this W92F obelin determined at 1.72 Angstrom resolution shows that there is no significant change in the dimensions of the active site between WT obelin (recombinant Ca2+-regulated photoprotein from Obelia longissima) and the mutant. It is proposed that the bioluminescence spectral shift results from removal of a hydrogen bond from the indole of W92 nearby a hydroxyl belonging to the 6-phenyl substituent of the substrate coelenterazine. Propagation of fbis change through a conjugated bond system in the excited state of the product coelenteramide affects the coupling of the N1-position and the hydrogen-bonded Y138. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

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

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19.

Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator [Text] / B. A. Illarionov [et al.] // Methods Enzymol. - 2000. - Vol. 305. - P223-249. - Cited References: 58 . - ISSN 0076-6879

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology
Рубрики:
PHOTOPROTEIN OBELIN
   MESSENGER-RNA
   CA-2+-ACTIVATED PHOTOPROTEIN
   DIRECTED MUTAGENESIS
   SEQUENCE-ANALYSIS
   HYDROID OBELIA
   AEQUORIN
   PROTEIN
   BIOLUMINESCENCE
   LUMINESCENCE

Держатели документа:
Russian Acad Sci, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
Univ Washington, Friday Harbor Labs, Friday Harbor, WA 98250 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Illarionov, B.A.; Frank, L.A.; Illarionova, V.A.; Bondar, V.S.; Vysotski, E.S.; Blinks, J.R.

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20.

Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the bioluminescent hydroid Obelia longissima [Text] / E. S. Vysotski [et al.] // Acta Crystallogr. Sect. D-Biol. Crystallogr. - 1999. - Vol. 55. - P1965-1966, DOI 10.1107/S0907444999011828. - Cited References: 23 . - ISSN 0907-4449

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Biophysics + Crystallography
Рубрики:
AEQUORIN
LUCIFERASE
LIGHT
Аннотация: Crystals of recombinant obelin, the Ca2+-regulated photoprotein from the marine hydroid Obelia longissima, have been grown from sodium citrate solutions. Crystals grow as hexagonal light-yellow rods (0.1 x 0.1 x 1.0 mm) which diffract to beyond 1.8 Angstrom with synchrotron radiation of 1.0 Angstrom wavelength. The crystals have a primitive hexagonal lattice with unit-cell parameters a = 81.55, c = 86.95 Angstrom. The asymmetric unit contains two molecules. This represents the successful preparation of single crystals of a photoprotein obelin which have promising diffraction properties.

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

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

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