• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Real time measurement of a switching mechanism in a signal transduction system on the basis of three dimensional structure.

Research Project

Project/Area Number 09680654
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionHimeji Institute of Technology

Principal Investigator

NAKAGAWA Masashi  Himeji Institute of Technology, Department of Life Science, assistant researcher, 理学部, 助手 (00212085)

Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Keywordsvision / rhodopsin / G-protein / fluorescence energy transfer / signal transduction / real time measurement / fluorescence label / photointermediates / G蛋白質 / 蛋白質間相互作用
Research Abstract

Development of a high sensitive and high time-resolved fluoresce measurement system
We developed a new fluorescence measurement system with high-time resolution and high sensitivity. This system allowed us to measure the fluorescence of a sample even contained a photolabile molecule, rhodopsin, with millisecond time resolution. We measured the transient fluorescence change of octopus rhodopsin, labeled by fluorescence dye specifically at Cys345 of the C terminal, upon light activation. It was found that the fluorescence of the dye changed with the time constant of about 1 msec. On the other hand, the absorptional change, reflecting the micro environment around the retinal chromophore, was completed within 100 msec. These results indicated that the conformational change around C-terminal took place after the conformational change around a retinal. It was suggested that the C-terminal conformational change may be involved in the interaction with G-protein.
The real-time measurement of the activation of G-protein by using fluorescence labeled GTP analogue
So far, it has been known that the intrinsic fluorescence of G-protein can monitor its active or inactive state. However, the detection is quite difficult under the circumstance mixed with other proteins like receptor. To circumvent this problem, we tried to detect the selective fluorescence from the only GTP bound to G-protein by using the fluorescent GTP analogue (mant (N-Methyl-3'-O-anthranoyl)-GTP, mant-GTPyS) on the basis of the fluorescence energy transfer principle. Consequently, we succeeded in recording, in real time, the GDP/GTP exchange reaction process upon photo-activation of rhodopsin. Furthermore, this method can also monitor GTP hydrolyzed process, because G-protein has so low affinity for the hydrolyzed mant-GDP that it rapidly release from G-protein, accompanied by the fluorescence decaying.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Nakagawa,M: "A novel photointermediate of octopus rhodopsin activates its G-protein." FEBS Lett.436. 259-262 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kikkawa,S: "A Novel Rhodopsin Kinase in Octopus Photoreceptor Possesses a Pleckstrin Homology Domain and is activated by G protein βγ Subunits." J.Biol.Chem.273. 7441-7447 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Iwasa,T: "NMR and UV-VIS absorption spectroscopic studies on fluorinated octopus rhodopsin and its photoproducts." J.Phys.Chem.A. 102. 5602-5610 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kobayasi,T.: "Femtosecond Spectroscopy of Halorhodopsin and Rhodopsin in a Broad Spectral Range of 410-1000nm." J.Phys.Chem.B. 102. 272-280 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakagawa,M: "Identification of Two Palmitoyl Groups in Octopus Rhodopsin." Photochem.Photobiol.65. 187-191 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakagawa,M: "Light-induced Protein Conformational Changes in the Photolysis of Octopus Rhodopsin." Biophysical.J.72. 2320-2328 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nishimura,S.: "structural dynamics of Waters and the Peptide Backbone around the Schiff Based Associated with the Light-activated Process of Octopus Rhodopsin." Biochemistry. 36. 864-870 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakagawa, M: "A novel photointermediate of optopus rhodopsin activates its G-protein." FEBS Lett.436. 259-262 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kikkawa, S: "A Novel Rhodopsin Kinase in octopus Photoreceptor Possesses a Pleckstrin Homology Domain and is activated by G protein betagamma Subunits." J.Biol.Chem.273. 7441-7447 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Iwasa, T.: "NMR and UV-VIS absorption spectroscopic studies on fluorinated octopus rhodopsin and its photoproducts." J.Phys.Chem.A102. 5602-5610 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kobayashi, T.: "Femtosecond Spectroscopy of Halorhodopsin and Rhodopsin in a Broad Spectral Range of 400-1000nm." J.Phys.Chem.B102. 272-280 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakagawa, M.: "Identification of Two Palmitoyl Groups in Octopus Rhodopsin." Photochem.Photobiol.65. 187-191 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakagawa, M.: "Light-induced Protein Conformational Changes in the Photolysis of Octopus Rhodopsin." Biophysical J.72. 2320-2328 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nishimura, S.: "Structural Dynamics of Waters and the Peptide Backbone around the Schiff Base associated with the Light-activated Process of Octopus Rhodopsin." Biochemistry. 36. 864-870 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nakagawa,M.: "A novel photointermediate of octopus rhodopsin activates its G-protein." FEBS Lett.436. 259-262 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kikkawa,S.: "A Novel Rhodopsin Kinase in Octopus Photoreceptor Possesses a Pleckstrin Homology Domain and is activated by G protein βγ Subunits." J.Biol.Chem.273. 7441-7447 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Iwasa,T.: "NMR and UV-VIS absorption spectroscopic studies on fluorinated octopus rhodopsin and its photoproducts." J.Phys.Chem.A. 102. 5602-5610 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kobayashi,T.: "Femtosecond Spectroscopy of Halorhodopsin and Rhodopsin in a Broad Spectral Range of 400-1000 nm." J.Phys.Chem.B. 102. 272-280 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nakagawa, M.: "Identification of Two Palmitoyl Groups in Octopus Rhodopsin" Photochem. Photobiol.65. 187-191 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Nakagawa, M.: "Light-Induced Protein Conformational Changes in the Photolysis of Octopas Rhodopsin" Biophys. J.72. 2320-2328 (1997)

    • Related Report
      1997 Annual Research Report

URL: 

Published: 1997-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi