Study of dynamic characteristic of muscle proteins by nanosecond time-resolved energy transfer measurements.

• Project/Area Number: 10680629
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: Fukui University
• Principal Investigator: MIKI Masao Faculty of Engineering, Fukui University, Applied Chemistry and Biotechnology, Professor, 工学部, 教授 (30242580)
• Project Period (FY): 1998 – 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥500,000 (Direct Cost: ¥500,000); Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
• Keywords: nanosecond time-resolved / Fluorescence energy transfer / muscle proteins / regulation of muscle contraction / troponin / tropomyosin / 筋タンパク

Research Abstract

The purpose of this project is studying of dynamic characteristics of muscle proteins by nanosecond time-resolved energy transfer measurements. We repaired the nanosecond time-resolved fluorometer which was transferred from Department of Physics, Nagoya University. On the other hand, we prepared mutants tropomyosin and troponin I by expressing in Escherichia coli for FRET measurements. We studied the spatial relationships between fluorescent probes attached to specific residues on actin, tropomyosin and troponin and then measured the distance changes between fluorescent probes in response to a change in CaィイD12+ィエD1 concentration. Through these studies, we obtained several important data for understanding the regulation mechanism by troponin-tropomyosin. Tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory. The C-terminal domain of troponin I moves to the outer domain of actin during inhibition, while the C-terminal domain of troponin C does not move much. The CaィイD12+ィエD1-induced changes in tropomyosin-troponin complex seems to occur only when F-actin is present, suggesting that a stable complex formation of troponin I with the outer domain of F-actin upon removal of CaィイD12+ィエD1 is very important event during inhibition. Then we have introduced a new model for the CaィイD12+ィエD1-mediated regulation of tropomyosin-troponin in skeletal muscle in replace to the steric blocking theory. Although we got several important results for revealing the regulation mechanism, we do not apply the lifetime measurements to analyze the fluorescence energy transfer. Therefore, we have to continue this project to develop the methods of fluorescence energy transfer.

Research Products

• [Publications] Miki, M. et al.: "Ca^<2+> -induced distance change between points on actin and troponin in skeletal muscle thin filaments estimated by fluorescence energy transfer spectroscopy"J. Biochem. 123. 324-331 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Miki, M. et al.: "Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments : Does tropomyosin move?"J. Biochem. 123. 1104-1111 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hai, H. et al.: "Conformational Changes of the troponin-tropomyosin complex on F-actin observed by fluorescence resonance energy transfer measurements"J. Fluorescence. (in press). (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Miki, M.: "Structural changes between regulatory proteins and actin : A regulation model by tropomyosin-troponin based on FRET measurements. In Molecular Interactions of Actin (dos Remedios, C. G. and Thomas, D. D., eds)"Springer Verlag, Heidelberg (in press). (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Miki, M., Kobayashi, T., Kimura, H., Hagiwara, A., Hai, H., and Maeda, Y.: "CaィイD12+ィエD1-induced distance change between points on actin and troponin in skeletal muscle thin filaments estimated by fluorescence energy transfer spectroscopy"J. Biochem.. 123. 324-331 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Miki, M., Miura, T., Sano, K., Kimura, H., Kondo, H., Ishida, H., and Maeda, Y.: "Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments: Does tropomyosin move?"J. Biochem.. 123. 1104-1111 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hai, H., Miura, T., Kobayashi, T., Maeda, Y., and Miki, M.: "Conformational Changes of the troponin-tropomyosin complex on F-actin observed by fluorescence resonance energy transfer measurements."J. Fluorescence. (in press). (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Miki, M.: "Structural changes between regulatory proteins and actin: A regulation model by tropomyosin-troponin based on FRET measurements in Molecular Interactions of Actin (dos Remedios, C. G. and Thomas, D. D., eds)"Springer Verlag, Heidelberg (in press).
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Miki,M.et al.: "Ca^<2+>-induced distance change between points on actin and troponin in skeletal muscle thin filaments estimated by fluorescence energy transfer spectroscopy."J.Biohem. 123. 324-331 (1998)
• [Publications] Miki,M.et al.: "Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments: Does tropomyosin move?."J.Biohem. 123. 1104-1111 (1998)
• [Publications] Hai,H.et al.: "Conformational Changes of the troponin-tropomyosin complex on F-actin observed by fluorescence resonance energy transfer measurements."J.Fluorescence. (in press). (2000)
• [Publications] Miki,M.: "Structural changes between regulatory proteins and actin: A regulation model by tropomyosin-troponin based on FRET measurements in Molecular Interactions of Actin (dos Remedios, C.G. and Thomas, D.D., eds)"Springer Verlag,Heidelberg (in press). (2000)