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Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering

View Article: PubMed Central - PubMed

ABSTRACT

Using small-angle X-ray scattering (SAXS), we have studied the initial stage (nucleation and oligomerization) of actin polymerization induced by raising temperature in a stepwise manner from 1°C to 30°C at low ionic strength (4.0 mg ml−1 actin in G-buffer). The SAXS experiments were started from the mono-disperse G-actin state, which was confirmed by comparing the scattering pattern in q- and real space with X-ray crystallographic data. We observed that the forward scattering intensity I(q → 0), used as an indicator for the extent of poly-merization, began to increase at ∼14°C for Mg-actin and ∼20°C for Ca-actin, and this critical temperature did not depend on the nucleotide species, i.e., ATP or ADP. At the temperatures higher than ∼20°C for Mg-actin and ∼25°C for Ca-actin, the coherent reflection peak, which is attributed to the helical structure of F-actin, appeared. The pair-distance distribution functions, p(r), corresponding to the frequency of vector lengths (r) within the molecule, were obtained by the indirect Fourier transformation (IFT) of the scattering curves, I(q). Next, the size distributions of oligomers at each temperature were analyzed by fitting the experimentally obtained p(r) with the theoretical p(r) for the helical and linear oligomers (2–13mers) calculated based on the X-ray crystallographic data. We found that p(r) at the initial stage of polymerization was well accounted for by the superposition of monomer, linear/helical dimers, and helical trimer, being independent of the type of divalent cations and nucleotides. These results suggest that the polymerization of actin in G-buffer induced by an increase in temperature proceeds via the elongation of the helical trimer, which supports, in a structurally resolved manner, a widely believed hypothesis that the polymerization nucleus is a helical trimer.

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Temperature dependence of SAXS data for Ca-ATP and Mg-ATP actins.(a) and (c): Scattering intensities I(q), and (b) and (d): Corresponding pair-distance distribution functions, p(r), for 4.0 mg ml−1 Ca-ATP (a), (c) and Mg-ATP (b), (d) actin solutions as a function of temperature. Arrows in (a) and (c) highlight the appearance of the orientationally averaged fiber reflection peak at q∼1.15 nm−1 (see text).
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f2-6_1: Temperature dependence of SAXS data for Ca-ATP and Mg-ATP actins.(a) and (c): Scattering intensities I(q), and (b) and (d): Corresponding pair-distance distribution functions, p(r), for 4.0 mg ml−1 Ca-ATP (a), (c) and Mg-ATP (b), (d) actin solutions as a function of temperature. Arrows in (a) and (c) highlight the appearance of the orientationally averaged fiber reflection peak at q∼1.15 nm−1 (see text).

Mentions: Figure 2 shows the temperature dependence of the scattering intensity I(q) and the corresponding p(r) function in the presence of ATP with either Ca2+ (Figs. 2a, b) or Mg2+ (Figs. 2c, d). The forward scattering intensity, I(q→0), abruptly increased at a certain temperature, demonstrating the initiation of polymerization. We call this temperature an onset temperature of polymerization T* (see arrows in Figs. 2a, c). The reflection peak, which appeared distinctly at q=1.15 nm−1, corresponds to the d-spacing of ca. 5.5 (=2π/1.15) nm. This can be recognized as a manifestation of orientationally averaged fiber diffraction peaks corresponding to the subunit axial translation, which are the so-called sixth- and seventh-layer lines known as the major characteristics of the established F-actin structure, i.e., a 13/6 symmetry. The peak at q∼2.3 nm−1, corresponding to the distance between the neighboring subunits along the F-actin filament, could also be seen, though somewhat vaguely.


Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering
Temperature dependence of SAXS data for Ca-ATP and Mg-ATP actins.(a) and (c): Scattering intensities I(q), and (b) and (d): Corresponding pair-distance distribution functions, p(r), for 4.0 mg ml−1 Ca-ATP (a), (c) and Mg-ATP (b), (d) actin solutions as a function of temperature. Arrows in (a) and (c) highlight the appearance of the orientationally averaged fiber reflection peak at q∼1.15 nm−1 (see text).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036667&req=5

f2-6_1: Temperature dependence of SAXS data for Ca-ATP and Mg-ATP actins.(a) and (c): Scattering intensities I(q), and (b) and (d): Corresponding pair-distance distribution functions, p(r), for 4.0 mg ml−1 Ca-ATP (a), (c) and Mg-ATP (b), (d) actin solutions as a function of temperature. Arrows in (a) and (c) highlight the appearance of the orientationally averaged fiber reflection peak at q∼1.15 nm−1 (see text).
Mentions: Figure 2 shows the temperature dependence of the scattering intensity I(q) and the corresponding p(r) function in the presence of ATP with either Ca2+ (Figs. 2a, b) or Mg2+ (Figs. 2c, d). The forward scattering intensity, I(q→0), abruptly increased at a certain temperature, demonstrating the initiation of polymerization. We call this temperature an onset temperature of polymerization T* (see arrows in Figs. 2a, c). The reflection peak, which appeared distinctly at q=1.15 nm−1, corresponds to the d-spacing of ca. 5.5 (=2π/1.15) nm. This can be recognized as a manifestation of orientationally averaged fiber diffraction peaks corresponding to the subunit axial translation, which are the so-called sixth- and seventh-layer lines known as the major characteristics of the established F-actin structure, i.e., a 13/6 symmetry. The peak at q∼2.3 nm−1, corresponding to the distance between the neighboring subunits along the F-actin filament, could also be seen, though somewhat vaguely.

View Article: PubMed Central - PubMed

ABSTRACT

Using small-angle X-ray scattering (SAXS), we have studied the initial stage (nucleation and oligomerization) of actin polymerization induced by raising temperature in a stepwise manner from 1°C to 30°C at low ionic strength (4.0 mg ml−1 actin in G-buffer). The SAXS experiments were started from the mono-disperse G-actin state, which was confirmed by comparing the scattering pattern in q- and real space with X-ray crystallographic data. We observed that the forward scattering intensity I(q → 0), used as an indicator for the extent of poly-merization, began to increase at ∼14°C for Mg-actin and ∼20°C for Ca-actin, and this critical temperature did not depend on the nucleotide species, i.e., ATP or ADP. At the temperatures higher than ∼20°C for Mg-actin and ∼25°C for Ca-actin, the coherent reflection peak, which is attributed to the helical structure of F-actin, appeared. The pair-distance distribution functions, p(r), corresponding to the frequency of vector lengths (r) within the molecule, were obtained by the indirect Fourier transformation (IFT) of the scattering curves, I(q). Next, the size distributions of oligomers at each temperature were analyzed by fitting the experimentally obtained p(r) with the theoretical p(r) for the helical and linear oligomers (2–13mers) calculated based on the X-ray crystallographic data. We found that p(r) at the initial stage of polymerization was well accounted for by the superposition of monomer, linear/helical dimers, and helical trimer, being independent of the type of divalent cations and nucleotides. These results suggest that the polymerization of actin in G-buffer induced by an increase in temperature proceeds via the elongation of the helical trimer, which supports, in a structurally resolved manner, a widely believed hypothesis that the polymerization nucleus is a helical trimer.

No MeSH data available.


Related in: MedlinePlus