Limits...
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.


Evaluation of the oligomer distribution by fitting experimental p(r) with the linear combination of simulated p(r) for Mg-ATP-actin.(a) and (c): The experimentally obtained p(r) functions (open circles) and the fitting curves (red lines) obtained by the linear combination of simulated p(r)s for helical (a) or linear and helical (c) oligomer models for Mg-ATP-actin. In each figure, two red lines were drawn for the range in which all the fitting results obtained from 100 trials were included. (The red lines were depicted as a single line when the two lines were very close.) The numerical values in (a) and (c) are the range of R2 obtained by the maximum and the minimum values from 100 trials. (b) and (d): The histograms of oligomers obtained from the best-fit results using helical (b) or linear and helical (d) oligomer models, in which the error bar indicates the range between 90 percentile and 10 percentile values obtained from 100 trials.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036667&req=5

f7-6_1: Evaluation of the oligomer distribution by fitting experimental p(r) with the linear combination of simulated p(r) for Mg-ATP-actin.(a) and (c): The experimentally obtained p(r) functions (open circles) and the fitting curves (red lines) obtained by the linear combination of simulated p(r)s for helical (a) or linear and helical (c) oligomer models for Mg-ATP-actin. In each figure, two red lines were drawn for the range in which all the fitting results obtained from 100 trials were included. (The red lines were depicted as a single line when the two lines were very close.) The numerical values in (a) and (c) are the range of R2 obtained by the maximum and the minimum values from 100 trials. (b) and (d): The histograms of oligomers obtained from the best-fit results using helical (b) or linear and helical (d) oligomer models, in which the error bar indicates the range between 90 percentile and 10 percentile values obtained from 100 trials.

Mentions: We attempted to describe the experimental profiles of p(r) in solution at T*≤T/°C≤TF with those calculated from crystallographic data as shown in Fig. 4. Figure 6 shows that the p(r) data for Ca-ATP-actin can be explained by the weighted sum of the theoretical p(r) functions for a monomer and different oligomers. Figures 6a and b present the results of the fitting procedures using a monomer and helical oligomers, excluding linear oligomers, and Figs. 6c and d provide those with the mixture of monomer, helical oligomers, and the postulated linear oligomers. These results show that the simulation was not substantially improved by including the contribution of linear oligomers (judging from the value of R2 shown in each figure), implying that the characteristic features of the experimentally obtained p(r)s can be interpreted as the mixture of helical oligomers and a monomer (Fig. 6b). The results of the similar analysis for Mg-ATP-actin, Ca-ADP-actin, and Mg-ADP-actin, are shown in Fig. 7 and Supplementary Figs. S2 and S3, respectively.


Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering
Evaluation of the oligomer distribution by fitting experimental p(r) with the linear combination of simulated p(r) for Mg-ATP-actin.(a) and (c): The experimentally obtained p(r) functions (open circles) and the fitting curves (red lines) obtained by the linear combination of simulated p(r)s for helical (a) or linear and helical (c) oligomer models for Mg-ATP-actin. In each figure, two red lines were drawn for the range in which all the fitting results obtained from 100 trials were included. (The red lines were depicted as a single line when the two lines were very close.) The numerical values in (a) and (c) are the range of R2 obtained by the maximum and the minimum values from 100 trials. (b) and (d): The histograms of oligomers obtained from the best-fit results using helical (b) or linear and helical (d) oligomer models, in which the error bar indicates the range between 90 percentile and 10 percentile values obtained from 100 trials.
© Copyright Policy
Related In: Results  -  Collection

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

f7-6_1: Evaluation of the oligomer distribution by fitting experimental p(r) with the linear combination of simulated p(r) for Mg-ATP-actin.(a) and (c): The experimentally obtained p(r) functions (open circles) and the fitting curves (red lines) obtained by the linear combination of simulated p(r)s for helical (a) or linear and helical (c) oligomer models for Mg-ATP-actin. In each figure, two red lines were drawn for the range in which all the fitting results obtained from 100 trials were included. (The red lines were depicted as a single line when the two lines were very close.) The numerical values in (a) and (c) are the range of R2 obtained by the maximum and the minimum values from 100 trials. (b) and (d): The histograms of oligomers obtained from the best-fit results using helical (b) or linear and helical (d) oligomer models, in which the error bar indicates the range between 90 percentile and 10 percentile values obtained from 100 trials.
Mentions: We attempted to describe the experimental profiles of p(r) in solution at T*≤T/°C≤TF with those calculated from crystallographic data as shown in Fig. 4. Figure 6 shows that the p(r) data for Ca-ATP-actin can be explained by the weighted sum of the theoretical p(r) functions for a monomer and different oligomers. Figures 6a and b present the results of the fitting procedures using a monomer and helical oligomers, excluding linear oligomers, and Figs. 6c and d provide those with the mixture of monomer, helical oligomers, and the postulated linear oligomers. These results show that the simulation was not substantially improved by including the contribution of linear oligomers (judging from the value of R2 shown in each figure), implying that the characteristic features of the experimentally obtained p(r)s can be interpreted as the mixture of helical oligomers and a monomer (Fig. 6b). The results of the similar analysis for Mg-ATP-actin, Ca-ADP-actin, and Mg-ADP-actin, are shown in Fig. 7 and Supplementary Figs. S2 and S3, respectively.

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.