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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.

No MeSH data available.


The model-free cross-section structure analysis of actin oligomers.Cross-section pair-distance distribution functions pc(r) of actin oligomers for Ca-ATP at 23°C. Also shown are the simulated pc(r) for crystal structures of the elongated helical (H13) and linear (L7) aggregates. For comparison, we additionally plotted the simulated pc(r) for a monomer and short-chain oligomers, L2 and H4.
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f8-6_1: The model-free cross-section structure analysis of actin oligomers.Cross-section pair-distance distribution functions pc(r) of actin oligomers for Ca-ATP at 23°C. Also shown are the simulated pc(r) for crystal structures of the elongated helical (H13) and linear (L7) aggregates. For comparison, we additionally plotted the simulated pc(r) for a monomer and short-chain oligomers, L2 and H4.

Mentions: Figure 8 displays the pair-distance distribution functions of the cross-section, pc(r), calculated from the equation (3) (see Materials and Methods), for Ca-ATP-actin at 23°C, together with pc(r) calculated for the crystal structures of helical and linear oligomers. With increasing XM, the features of pc(r) became markedly closer to those predicted for the long helical structure. When XM= 0.6 was assumed, the experimental pc(r) nicely fitted the theoretical pc(r) for H13. On the other hand, according to the fitting analysis (Fig. 6), the monomer weight fraction for Ca-ATP-actin at 23°C was ∼0.4. This apparent discrepancy could be explained by the contribution from the co-existing short oligomers such as H4 (Fig. 6b). For simplicity, in this section we assumed the mixture of a monomer and a single type of a long oligomer. Therefore, if we perform the cross-section structure analysis including short oligomers like L2 and H4 (gray curves in Fig. 8) in addition to a monomer and H13 as shown in Fig. 6b, this apparent discrepancy may be eliminated.


Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering
The model-free cross-section structure analysis of actin oligomers.Cross-section pair-distance distribution functions pc(r) of actin oligomers for Ca-ATP at 23°C. Also shown are the simulated pc(r) for crystal structures of the elongated helical (H13) and linear (L7) aggregates. For comparison, we additionally plotted the simulated pc(r) for a monomer and short-chain oligomers, L2 and H4.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036667&req=5

f8-6_1: The model-free cross-section structure analysis of actin oligomers.Cross-section pair-distance distribution functions pc(r) of actin oligomers for Ca-ATP at 23°C. Also shown are the simulated pc(r) for crystal structures of the elongated helical (H13) and linear (L7) aggregates. For comparison, we additionally plotted the simulated pc(r) for a monomer and short-chain oligomers, L2 and H4.
Mentions: Figure 8 displays the pair-distance distribution functions of the cross-section, pc(r), calculated from the equation (3) (see Materials and Methods), for Ca-ATP-actin at 23°C, together with pc(r) calculated for the crystal structures of helical and linear oligomers. With increasing XM, the features of pc(r) became markedly closer to those predicted for the long helical structure. When XM= 0.6 was assumed, the experimental pc(r) nicely fitted the theoretical pc(r) for H13. On the other hand, according to the fitting analysis (Fig. 6), the monomer weight fraction for Ca-ATP-actin at 23°C was ∼0.4. This apparent discrepancy could be explained by the contribution from the co-existing short oligomers such as H4 (Fig. 6b). For simplicity, in this section we assumed the mixture of a monomer and a single type of a long oligomer. Therefore, if we perform the cross-section structure analysis including short oligomers like L2 and H4 (gray curves in Fig. 8) in addition to a monomer and H13 as shown in Fig. 6b, this apparent discrepancy may be eliminated.

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.