2003 Fiscal Year Final Research Report Summary
X-ray diffraction analysis of structural charges of regulatory proteins and tins in muscle contraction.
| Project/Area Number |
13480220
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
WAKABAYASHI Katsuzo Osaka University, Graduate School of Engineering Science, Professor, 大学院・基礎工学研究科, 教授 (00029521)
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| Co-Investigator(Kenkyū-buntansha) |
UENO Yutaka National Institute of Advanced Industrial Science and Technology, Principal Investigator, 脳神経情報部門, 主任研究員 (60356558)
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| Project Period (FY) |
2001 – 2003
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| Keywords | muscle contraction / regulatory mechanism / troponin / thin actin filament / X-ray diffractions / synchrotron radiation / time-resolved X-ray diffraction / tropomyoin |
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| Research Abstract |
Structural changes of the regulatory proteins (troponin and tropomyosin) and the thin actin filaments occurring in the muscle contraction process have been measured by using an intense synchrotron X-ray diffraction, and their data were analyzed. The obtained results are as follows.
(1)Time-resolved X-ray diffraction with a CCD-based 2D detector in the process of isometric force development of muscle at the full overlap length of thin and thick filaments showed that the first troponin-associated meridional reflection (TN1) increased its intensity at the onset of stimulation to ca. 140% and then decreased it together with the development of force, reaching the level below the resting value. The TN2 intensity decreased in parallel with the development of tension but the TN3 intensity increased and run ahead the tension. On activation of muscle at the non-overlap length, the intensity of TN1 increased by ca. 40% on the onset of stimulation but stayed at the ca. 140% level during the plateau
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phase of force. However, their radial reflection width increased with the development of force. After the correction of the radial width, the TN1 intensity decayed and stayed almost at the resting level after an initial increase. The modeling analysis of the intensity changes of TN1-TN4 suggested that on activation the elongated domain moved relative to the globular domain of a TN molecule to increase the overlap between the two domains and during the plateau level of force development the overlap between the two domains decreased toward the resting state.
The results indicates that the structural changes of the TN molecules which are related to the regulatory mechanism in muscle contraction are occurring in the two processes, namely on activation and during force generation.
(2)With the use of a low emittance synchrotron beam, the close relationship between the extensibility and twisting changes of the thin actin filaments was clarified by investigating the first actin-based layer line in the 2D X-ray diffraction patterns from a contracting muscle by the application of stretch. The relative spacing change of the first layer line was ca. 0.90% during stretch applied to contracting muscle and was -1.38% on activation of the overstretched muscle, confirming that the extensibility is closely related with the nature of the twisting of the left-and right-handed genetic helices. The helical symmetry of the actin filament altered from an 80 units/37 turns symmetry in the resting state through a 106 units/49 turns on activation to a 54 units/25 turns symmetry in the force generating state. These results clearly indicate that the thin actin filament itself include the on-off-states, operating as the Ca2+ switch and during contraction the extensibility of the actin filaments is directly related to the extention of the elastic element in muscle.
(3)The 2D X-ray diffraction patterns with a high-spatial resolution were taken during contraction, and the intensities of the actin-based layer lines were measured upto thel.37 nm reflection corresponding to the third order actin meridional reflection. Using the higher-angle intensity data of the layer lines above the 7.1 nm, the modeling analysis of the actin filament including the core domain of troponin molecules was performed by starting from the Holmes' atomic filament model (1990). In the modeling the actin monomer in the filament was subdivided into 16 segments without destroying the secondary structures and they were moved as a rigid body. The disposition and arrangement of the troponin core domains on the actin filament were searched to obtain the minimum value of the R-factor. The best-fit resting model of the actin filament was determined with some modification of the Holmes' model. The troponin core domain bound over the subdomains 1 and 2 of actin in the filament. The results indicate that small movements of each four subdomains in the actin monomer as well as the disposition of the troponin core domain portion take place during contraction. The largest movement was the subdomain 2; it moved toward the filament axis by ca. 0.5 nm. The TNC part moved toward the outside of the actin filament. Less
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