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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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X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length. (A) A comparison of diffraction patterns in the low- to medium-angle region between the relaxed and the activated states, and (B) a comparison of those in the medium- to high-angle region between them. The meridional axis (M) is coincided. E is the equatorial axis. The letter T with an index of the 384 Å repeat denotes the troponin-associated meridional reflections and the letter A with the axial spacing in Ångstrom unit, the representative thin filament-based layer lines.
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f1-6_13: X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length. (A) A comparison of diffraction patterns in the low- to medium-angle region between the relaxed and the activated states, and (B) a comparison of those in the medium- to high-angle region between them. The meridional axis (M) is coincided. E is the equatorial axis. The letter T with an index of the 384 Å repeat denotes the troponin-associated meridional reflections and the letter A with the axial spacing in Ångstrom unit, the representative thin filament-based layer lines.

Mentions: Figure 1 shows the low- to moderate-angle (A) and the moderate- to high-angle (B) X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length by using synchrotron X-rays, in which the diffraction patterns in the relaxed state and upon activation are compared with the meridional axes coincident. The layer line reflections from the thick and the thin filaments are observed with different periodicities, and the representative reflections from the thin filaments are marked by the letters T and A in Fig. 1. Figure 2 shows the comparison of the intensity data of the thin filament-based layer lines in the relaxed state and upon activation of non-filament overlapped muscle. The intensity changes of the thin filament reflections occurring upon activation were much smaller than those during contraction of muscle at full-filament overlap length9. The first and the second thin filament-based layer lines exhibited distinct intensity changes upon activation; the first layer line intensity decreased by ∼39% and the second layer line intensity rose from nearly zero level at rest to ∼16% of the 59 Å layer line intensity. The 59 Å and the 51 Å layer lines, whose axial spacings correspond to the pitches of the left and the right-handed genetic (1-start) helices, exhibited a small increase in intensity upon activation: the intensity increment of the main peak of the 51 Å layer line was ∼22%, greater than that of the 59 Å layer line which was almost the same, and the main peak of the 51 Å layer line shifted slightly away from the meridian. The second peak intensity of the 29 Å layer line markedly decreased by ∼40%, and that of the 27 Å layer line, whose spacing corresponds to the axial repeat of actin subunits, shifted away from the meridian. The reciprocal intensity change of the first and the second layer lines implicates that the four-fold rotational symmetry nature of the thin filament structure is strengthened in the activated state, because the Bessel function J4 contributes to the second layer line and J2 contributes to the first layer line. There were also small intensity changes observed for other layer lines. The first and the second troponin-associated meridional reflections with a repeat distance of 384 Å increased their intensities but the intensity of the third one decreased upon activaion9,14. Since they were modulated due to a confounding sampling effect, they were not used for the present modeling. There is no interaction of myosin heads with actin filaments in the overstretched muscle, and thus small but distinct intensity changes of the layer lines upon activation are ascribed solely to the structural changes within the thin filaments by the calcium-regulation mechanism.


X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles
X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length. (A) A comparison of diffraction patterns in the low- to medium-angle region between the relaxed and the activated states, and (B) a comparison of those in the medium- to high-angle region between them. The meridional axis (M) is coincided. E is the equatorial axis. The letter T with an index of the 384 Å repeat denotes the troponin-associated meridional reflections and the letter A with the axial spacing in Ångstrom unit, the representative thin filament-based layer lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036664&req=5

f1-6_13: X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length. (A) A comparison of diffraction patterns in the low- to medium-angle region between the relaxed and the activated states, and (B) a comparison of those in the medium- to high-angle region between them. The meridional axis (M) is coincided. E is the equatorial axis. The letter T with an index of the 384 Å repeat denotes the troponin-associated meridional reflections and the letter A with the axial spacing in Ångstrom unit, the representative thin filament-based layer lines.
Mentions: Figure 1 shows the low- to moderate-angle (A) and the moderate- to high-angle (B) X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length by using synchrotron X-rays, in which the diffraction patterns in the relaxed state and upon activation are compared with the meridional axes coincident. The layer line reflections from the thick and the thin filaments are observed with different periodicities, and the representative reflections from the thin filaments are marked by the letters T and A in Fig. 1. Figure 2 shows the comparison of the intensity data of the thin filament-based layer lines in the relaxed state and upon activation of non-filament overlapped muscle. The intensity changes of the thin filament reflections occurring upon activation were much smaller than those during contraction of muscle at full-filament overlap length9. The first and the second thin filament-based layer lines exhibited distinct intensity changes upon activation; the first layer line intensity decreased by ∼39% and the second layer line intensity rose from nearly zero level at rest to ∼16% of the 59 Å layer line intensity. The 59 Å and the 51 Å layer lines, whose axial spacings correspond to the pitches of the left and the right-handed genetic (1-start) helices, exhibited a small increase in intensity upon activation: the intensity increment of the main peak of the 51 Å layer line was ∼22%, greater than that of the 59 Å layer line which was almost the same, and the main peak of the 51 Å layer line shifted slightly away from the meridian. The second peak intensity of the 29 Å layer line markedly decreased by ∼40%, and that of the 27 Å layer line, whose spacing corresponds to the axial repeat of actin subunits, shifted away from the meridian. The reciprocal intensity change of the first and the second layer lines implicates that the four-fold rotational symmetry nature of the thin filament structure is strengthened in the activated state, because the Bessel function J4 contributes to the second layer line and J2 contributes to the first layer line. There were also small intensity changes observed for other layer lines. The first and the second troponin-associated meridional reflections with a repeat distance of 384 Å increased their intensities but the intensity of the third one decreased upon activaion9,14. Since they were modulated due to a confounding sampling effect, they were not used for the present modeling. There is no interaction of myosin heads with actin filaments in the overstretched muscle, and thus small but distinct intensity changes of the layer lines upon activation are ascribed solely to the structural changes within the thin filaments by the calcium-regulation mechanism.

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