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X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles

View Article: PubMed Central - PubMed

ABSTRACT

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

No MeSH data available.


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Four subdomains of an actin molecule and division of 16 segments used in the modeling. (A) An actin molecule consists of four subdomains by Kabsch et al. (1990)15. Subdomains 1, 2, 3 and 4 are colored in red, orange, magenda and pink, respectively. (B) The division of the actin molecule into 16 segments. The sequential number of amino acid residues in the boundary between neighboring segments is written above the color bar and the corresponding segment number is written below the bar. (C) The surface display of the actin molecule devided into 16 segments. The number of the segments is indicated, and the color assignment for them is the same as in (B). The right panel is the view with the left one rotated by 180° around the vertical axis.
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f5-6_13: Four subdomains of an actin molecule and division of 16 segments used in the modeling. (A) An actin molecule consists of four subdomains by Kabsch et al. (1990)15. Subdomains 1, 2, 3 and 4 are colored in red, orange, magenda and pink, respectively. (B) The division of the actin molecule into 16 segments. The sequential number of amino acid residues in the boundary between neighboring segments is written above the color bar and the corresponding segment number is written below the bar. (C) The surface display of the actin molecule devided into 16 segments. The number of the segments is indicated, and the color assignment for them is the same as in (B). The right panel is the view with the left one rotated by 180° around the vertical axis.

Mentions: In the third step, the actin conformation was altered by changing the relative dispositions of four subdomains (Fig. 5A). The optimum dispositions of four subdomains together with the tropomyosin + TNT1 tail and the TN core domain were examined using the simulated annealing method22. The intensities of the first, second, 59 Å, 51 Å, 29 Å and 27 Å layer lines were used for fitting. The parameters for each actin subdomain were translations along x, y and z Cartesian coordinates, and those for the tropomyosin + TNT1 tail and the TN core domain were the same as in the above. Each subdomain was moved as a rigid body with a step of 0.2 Å within 3 Å so as to avoid collision. The azimuthal and radial parameters for the tropomyosin + TNT1 tail and the TN core domain were altered with 1° and 0.2 Å step, respectively. As the results, four subdomains of actin tended to move to close the nucleotide-binding cleft, making an actin molecule compact along the filament axis. Together with this subdomain movement in actin, the position of the tropomyosin + TNT1 tail and the orientation of the TN core domain altered, resulting in a reasonable fit to the observed low- to medium-angle intensity data. This fact indicates that movements of regulatory proteins alone on the Holmes F-actin model without changing the actin structure are insufficient to account for the low- to medium-angle intensity data. This conclusion may be in line with the simulations by Al-Khayat et al. (1995)23, Squire and Morris (1998)24 and Iwamoto et al. (2003)25. However, the fits to the high-angle intensities beyond the 29 Å layer line, which are dominated by the actin structure in F-actin, were still poor. The intensities of the high-angle layer lines could not be explained by global movements of four subdomains in the actin structure. We needed to introduce finer movements within the actin molecule to obtain a better fit to the high-angle intensity data.


X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles
Four subdomains of an actin molecule and division of 16 segments used in the modeling. (A) An actin molecule consists of four subdomains by Kabsch et al. (1990)15. Subdomains 1, 2, 3 and 4 are colored in red, orange, magenda and pink, respectively. (B) The division of the actin molecule into 16 segments. The sequential number of amino acid residues in the boundary between neighboring segments is written above the color bar and the corresponding segment number is written below the bar. (C) The surface display of the actin molecule devided into 16 segments. The number of the segments is indicated, and the color assignment for them is the same as in (B). The right panel is the view with the left one rotated by 180° around the vertical axis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036664&req=5

f5-6_13: Four subdomains of an actin molecule and division of 16 segments used in the modeling. (A) An actin molecule consists of four subdomains by Kabsch et al. (1990)15. Subdomains 1, 2, 3 and 4 are colored in red, orange, magenda and pink, respectively. (B) The division of the actin molecule into 16 segments. The sequential number of amino acid residues in the boundary between neighboring segments is written above the color bar and the corresponding segment number is written below the bar. (C) The surface display of the actin molecule devided into 16 segments. The number of the segments is indicated, and the color assignment for them is the same as in (B). The right panel is the view with the left one rotated by 180° around the vertical axis.
Mentions: In the third step, the actin conformation was altered by changing the relative dispositions of four subdomains (Fig. 5A). The optimum dispositions of four subdomains together with the tropomyosin + TNT1 tail and the TN core domain were examined using the simulated annealing method22. The intensities of the first, second, 59 Å, 51 Å, 29 Å and 27 Å layer lines were used for fitting. The parameters for each actin subdomain were translations along x, y and z Cartesian coordinates, and those for the tropomyosin + TNT1 tail and the TN core domain were the same as in the above. Each subdomain was moved as a rigid body with a step of 0.2 Å within 3 Å so as to avoid collision. The azimuthal and radial parameters for the tropomyosin + TNT1 tail and the TN core domain were altered with 1° and 0.2 Å step, respectively. As the results, four subdomains of actin tended to move to close the nucleotide-binding cleft, making an actin molecule compact along the filament axis. Together with this subdomain movement in actin, the position of the tropomyosin + TNT1 tail and the orientation of the TN core domain altered, resulting in a reasonable fit to the observed low- to medium-angle intensity data. This fact indicates that movements of regulatory proteins alone on the Holmes F-actin model without changing the actin structure are insufficient to account for the low- to medium-angle intensity data. This conclusion may be in line with the simulations by Al-Khayat et al. (1995)23, Squire and Morris (1998)24 and Iwamoto et al. (2003)25. However, the fits to the high-angle intensities beyond the 29 Å layer line, which are dominated by the actin structure in F-actin, were still poor. The intensities of the high-angle layer lines could not be explained by global movements of four subdomains in the actin structure. We needed to introduce finer movements within the actin molecule to obtain a better fit to the high-angle intensity data.

View Article: PubMed Central - PubMed

ABSTRACT

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

No MeSH data available.


Related in: MedlinePlus