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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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Searching parameters used in the model calculation. (A) Parameters for the optimal positioning of tropomyosin (white) around the fiber axis which is denoted by a star mark. r and θ are the radial distance and the azimuthal angle of tropomyosin around the fiber axis, respectively. The zero positions for the parameters are defined in the text. The positive direction of r is away from the fiber axis and the clockwise rotation is positive for θ. (B) Parameters used for the optimal positioning of the troponin core domain (left). r and θ are the radial distance and the azimuthal angle of the troponin core domain around the fiber axis, respectively. The parameters α, β and γ are the rotation angle around the x, y and z Cartesian coordinates, respectively. The N-terminus of the troponin core domain is located so that it connects with the C-terminus of TNT1 (right). The loci of these termini are depicted by red circles. The variable range of each parameter is given in Table1. F-actin is shown by blue balls, in which four subdomains of an actin molecule15 are colored by red (subdomain 1), orange (subdomain 2), magenda (subdomain 3) and pink (subdomain 4). Tropomyosin is shown by the two strands of white balls and the troponin three subunits are shown by light blue (TNC), green (TNI) and yellow balls (TNT).
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f3-6_13: Searching parameters used in the model calculation. (A) Parameters for the optimal positioning of tropomyosin (white) around the fiber axis which is denoted by a star mark. r and θ are the radial distance and the azimuthal angle of tropomyosin around the fiber axis, respectively. The zero positions for the parameters are defined in the text. The positive direction of r is away from the fiber axis and the clockwise rotation is positive for θ. (B) Parameters used for the optimal positioning of the troponin core domain (left). r and θ are the radial distance and the azimuthal angle of the troponin core domain around the fiber axis, respectively. The parameters α, β and γ are the rotation angle around the x, y and z Cartesian coordinates, respectively. The N-terminus of the troponin core domain is located so that it connects with the C-terminus of TNT1 (right). The loci of these termini are depicted by red circles. The variable range of each parameter is given in Table1. F-actin is shown by blue balls, in which four subdomains of an actin molecule15 are colored by red (subdomain 1), orange (subdomain 2), magenda (subdomain 3) and pink (subdomain 4). Tropomyosin is shown by the two strands of white balls and the troponin three subunits are shown by light blue (TNC), green (TNI) and yellow balls (TNT).

Mentions: The model calculations were systematically performed by the procedure as follows. In the first step, the TNT1 parts were arranged along tropomyosin molecules so that their C-termini can be connected to the N-termini of TNT2 parts (see Fig. 3B). At present there is no evidence that the TNT1 part, which is a calcium-insensitive anchor, is separated from the tropomyosin strands16. The optimal disposition of the tropomyosin molecule with the TNT1 part, which is located initially at the appropriate position, was searched by moving around the periphery of F-actin and radially (Fig. 3A) to obtain good fits of the calculated intensities of the first and the second layer lines to the observed intensities in the relaxed state. The range and the step sizes for the angular (θ) and radial (r) parameters are given in Table 1A. It was not possible to obtain a good fit to the observed intensities of these low-angle layer lines solely by the rotation of the tropomyosin + TNT1 tail around the initial F-actin model.


X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles
Searching parameters used in the model calculation. (A) Parameters for the optimal positioning of tropomyosin (white) around the fiber axis which is denoted by a star mark. r and θ are the radial distance and the azimuthal angle of tropomyosin around the fiber axis, respectively. The zero positions for the parameters are defined in the text. The positive direction of r is away from the fiber axis and the clockwise rotation is positive for θ. (B) Parameters used for the optimal positioning of the troponin core domain (left). r and θ are the radial distance and the azimuthal angle of the troponin core domain around the fiber axis, respectively. The parameters α, β and γ are the rotation angle around the x, y and z Cartesian coordinates, respectively. The N-terminus of the troponin core domain is located so that it connects with the C-terminus of TNT1 (right). The loci of these termini are depicted by red circles. The variable range of each parameter is given in Table1. F-actin is shown by blue balls, in which four subdomains of an actin molecule15 are colored by red (subdomain 1), orange (subdomain 2), magenda (subdomain 3) and pink (subdomain 4). Tropomyosin is shown by the two strands of white balls and the troponin three subunits are shown by light blue (TNC), green (TNI) and yellow balls (TNT).
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Related In: Results  -  Collection

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f3-6_13: Searching parameters used in the model calculation. (A) Parameters for the optimal positioning of tropomyosin (white) around the fiber axis which is denoted by a star mark. r and θ are the radial distance and the azimuthal angle of tropomyosin around the fiber axis, respectively. The zero positions for the parameters are defined in the text. The positive direction of r is away from the fiber axis and the clockwise rotation is positive for θ. (B) Parameters used for the optimal positioning of the troponin core domain (left). r and θ are the radial distance and the azimuthal angle of the troponin core domain around the fiber axis, respectively. The parameters α, β and γ are the rotation angle around the x, y and z Cartesian coordinates, respectively. The N-terminus of the troponin core domain is located so that it connects with the C-terminus of TNT1 (right). The loci of these termini are depicted by red circles. The variable range of each parameter is given in Table1. F-actin is shown by blue balls, in which four subdomains of an actin molecule15 are colored by red (subdomain 1), orange (subdomain 2), magenda (subdomain 3) and pink (subdomain 4). Tropomyosin is shown by the two strands of white balls and the troponin three subunits are shown by light blue (TNC), green (TNI) and yellow balls (TNT).
Mentions: The model calculations were systematically performed by the procedure as follows. In the first step, the TNT1 parts were arranged along tropomyosin molecules so that their C-termini can be connected to the N-termini of TNT2 parts (see Fig. 3B). At present there is no evidence that the TNT1 part, which is a calcium-insensitive anchor, is separated from the tropomyosin strands16. The optimal disposition of the tropomyosin molecule with the TNT1 part, which is located initially at the appropriate position, was searched by moving around the periphery of F-actin and radially (Fig. 3A) to obtain good fits of the calculated intensities of the first and the second layer lines to the observed intensities in the relaxed state. The range and the step sizes for the angular (θ) and radial (r) parameters are given in Table 1A. It was not possible to obtain a good fit to the observed intensities of these low-angle layer lines solely by the rotation of the tropomyosin + TNT1 tail around the initial F-actin model.

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