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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.


Comparison of the calculated intensities from the best-fit models and the observed intensities of the thin-filament-based layer lines. (A) The relaxed state and (B) the activated state. The layer line intensities of the 71 Å, 19.9 Å, 17,7 Å, 16.9 Å and 14.2 Å were mostly too weak to measure. They are denoted by small dotted curves. Rf for the eight layer lines in total was ∼11% in the relaxed state and ∼13% in the activated state.
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f7-6_13: Comparison of the calculated intensities from the best-fit models and the observed intensities of the thin-filament-based layer lines. (A) The relaxed state and (B) the activated state. The layer line intensities of the 71 Å, 19.9 Å, 17,7 Å, 16.9 Å and 14.2 Å were mostly too weak to measure. They are denoted by small dotted curves. Rf for the eight layer lines in total was ∼11% in the relaxed state and ∼13% in the activated state.

Mentions: Refinement simulation of the thin filaments was performed using the 16-segmented model of actin in the relaxed and activated states. Here the crystal structure of the TN core domain without Ca2+ ions (PDB ID: 1YV0) was commonly used for modeling because there was no significant change in the subunit arrangement in the absence and presence of Ca2+ ions4. Figure 6 shows the best-fit model of the thin filament in both states. It is evident that the best-fit model of the thin filament yielded nice fits to the observed X-ray diffraction data in each state (Figure 7); the Rf-factor was ∼11% in the relaxed model and ∼13% in the activated model for the observed layer line data. Although in the high-angle region the fits of intensity profiles near the meridian of the 29 Å, 27 Å and 13.7 Å layer lines were not satisfactory, the fits of the low- to the medium-angle data were excellent, and the discrepancy between the experimental and the calculated intensities seen in Fig. 4 was greatly improved. Note that when we attempted to fit the data in the activated state just by changing the orientation/disposition of the regulatory proteins using the best-fit model obtained in the relaxed state without changing the F-actin structure, it was unsuccessful. Our simulation results indicate that a conformational change of actin was inevitably needed to obtain satisfactory fits to all layer line intensities upon activation.


X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles
Comparison of the calculated intensities from the best-fit models and the observed intensities of the thin-filament-based layer lines. (A) The relaxed state and (B) the activated state. The layer line intensities of the 71 Å, 19.9 Å, 17,7 Å, 16.9 Å and 14.2 Å were mostly too weak to measure. They are denoted by small dotted curves. Rf for the eight layer lines in total was ∼11% in the relaxed state and ∼13% in the activated state.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036664&req=5

f7-6_13: Comparison of the calculated intensities from the best-fit models and the observed intensities of the thin-filament-based layer lines. (A) The relaxed state and (B) the activated state. The layer line intensities of the 71 Å, 19.9 Å, 17,7 Å, 16.9 Å and 14.2 Å were mostly too weak to measure. They are denoted by small dotted curves. Rf for the eight layer lines in total was ∼11% in the relaxed state and ∼13% in the activated state.
Mentions: Refinement simulation of the thin filaments was performed using the 16-segmented model of actin in the relaxed and activated states. Here the crystal structure of the TN core domain without Ca2+ ions (PDB ID: 1YV0) was commonly used for modeling because there was no significant change in the subunit arrangement in the absence and presence of Ca2+ ions4. Figure 6 shows the best-fit model of the thin filament in both states. It is evident that the best-fit model of the thin filament yielded nice fits to the observed X-ray diffraction data in each state (Figure 7); the Rf-factor was ∼11% in the relaxed model and ∼13% in the activated model for the observed layer line data. Although in the high-angle region the fits of intensity profiles near the meridian of the 29 Å, 27 Å and 13.7 Å layer lines were not satisfactory, the fits of the low- to the medium-angle data were excellent, and the discrepancy between the experimental and the calculated intensities seen in Fig. 4 was greatly improved. Note that when we attempted to fit the data in the activated state just by changing the orientation/disposition of the regulatory proteins using the best-fit model obtained in the relaxed state without changing the F-actin structure, it was unsuccessful. Our simulation results indicate that a conformational change of actin was inevitably needed to obtain satisfactory fits to all layer line intensities upon activation.

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.