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

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.

Keywords: X-ray scattering; actin trimer; oligomer distribution; solution structure.

Figure 1.

Particle characterization of G-actin by SAXS experiments. (a), Scattering intensity I(q), and (b), Corresponding pair-distance distribution function, p(r), of G-actin (Ca-ATP) at 4°C. p(r) refers to the spatial autocorrelation function of the electron density fluctuation of the protein in solution, which is obtained as inverse Fourier transformation of I(q). In the inset of (b), the three-dimensional structure of G-actin is presented by ribbon model. The pink solid curve shown in (b) represents (a) p(r) function calculated from the crystallographic data of G-actin (PDB code 1J6Z).

Figure 2.

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).

Figure 3.

Effect of temperature on polymerization of actin with different divalent cations and nucleotides. Relative average molecular weight of actin aggregates in solution, M̄_w(T)/M_w(T₀), plotted as a function of temperature determined from an extrapolated zero-angle scattering intensity I(q→0) is shown for Ca-ATP-actin (red), Ca-ADP-actin (orange), Mg-ATP-actin (blue), and Mg-ADP-actin (green). Circles and squares indicate, respectively, the non-fibrous and fibrous nature of actin oligomers judged from the absence or presence of the fiber reflection pattern in I(q). The temperature T*, at which polymerization starts, depends not on the nucleotide state, but on the divalent cation species.

Figure 4.

p(r) functions simulated from the model structures of helical and linear polymers. (a), p(r) simulated from the crystal structures of a helical polymer (dimer (H2)∼13mer (H13)) and a monomer. (b), p(r) simulated from the crystal structures of a linear polymer (dimer (L2)∼7mer (L7)) and a monomer. The actin concentration was 4.0 mg ml⁻¹. (c), Three-dimensional structures of various actin aggregates: a helical 13mer (H13) (left) and an assumed linear 7mer (L7) (right). The model structure was obtained from PDB data of the Holmes’ actin filament model. The structure of a linear polymer was produced from the helical structure (see text).

Figure 5.

Structural characterization of actin aggregates at the initial stage of polymerization. Analysis of the experimental p(r) at T∼T* assuming the mixture model of a monomer (M), a linear dimer (L2), a helical dimer (H2), and a helical trimer (H3): (a), Ca-ATP at 20°C, and (b), Mg-ATP at 13°C. (b) and (d), The examined 100 parameter sets for different weight fractions of H2, L2, and H3 (W_H2, W_L2 and W_H3) and their R² values obtained as a function of the monomer weight fraction (W_M). (a) and (c), The experimental p(r) (open black circles) and the fitting curve (pink solid line) based on the superposition of the simulated p(r) functions of M, L2, and H3 (sequential solid lines, orange, blue and red, respectively). In the insets of the panels (a) and (c), the experimental scattering intensity I(q) and the fitting curve using I(q) simulated from the crystal data are shown. This confirms simultaneous successful description of the scattering data in both q-space and a real space. Solid arrows indicate the minimal values of R². Dashed arrows indicate R² for the parameter sets that correspond to the panels (a) and (c).

Figure 6.

Evaluation of the oligomer distribution by fitting experimental p(r) with the linear combination of simulated p(r) for Ca-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 Ca-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.

Figure 7.

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.

Figure 8.

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