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.


Related in: MedlinePlus

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-actin25 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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036667&req=5

f1-6_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-actin25 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).

Mentions: Figure 1 shows a typical example of the SAXS experiments on G-actin, in which the data for Ca-ATP-actin at 1°C are presented. The scattering intensity I(q) (Fig. 1a) is given as a double-logarithmic plot. The pair distance distribution function, p(r) (Fig. 1b), was obtained by inverse Fourier transformation (IFT) of I(q). We found that the experimental p(r) for all G-actin species (Mg2+, Ca2+, ATP and ADP) in solution closely resembled p(r) calculated from the crystallographic data of ADP-G-actin25 without accounting for the contrast difference between hydrated and solvent water, which is shown by a solid line. On the other hand, it is known that the crystal structure of subdomain 2 of G-actin is slightly different between the ATP and ADP states26; besides, it was reported recently that the X-ray fiber diffraction of actin filaments showed that the angles between the subdomains 1, 2, and the subdomains 3, 4 in an actin monomer are slightly altered upon polymerization27. We calculated the p(r) functions using their reported structural data, but they were indistinguishable from those of ATP-G-actin (data not shown), implying that little structural alterations, such as small conformational changes in the subdomain orientation, are not clearly detectable in the p(r) analysis at the present resolution. Nevertheless, the data confirm the mono-dispersity of the solution within this resolution, implying that the G-actin solution at the initial stage of the following temperature-induced series of polymerization experiments was not contaminated with polymers.


Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering
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-actin25 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).
© Copyright Policy
Related In: Results  -  Collection

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

f1-6_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-actin25 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).
Mentions: Figure 1 shows a typical example of the SAXS experiments on G-actin, in which the data for Ca-ATP-actin at 1°C are presented. The scattering intensity I(q) (Fig. 1a) is given as a double-logarithmic plot. The pair distance distribution function, p(r) (Fig. 1b), was obtained by inverse Fourier transformation (IFT) of I(q). We found that the experimental p(r) for all G-actin species (Mg2+, Ca2+, ATP and ADP) in solution closely resembled p(r) calculated from the crystallographic data of ADP-G-actin25 without accounting for the contrast difference between hydrated and solvent water, which is shown by a solid line. On the other hand, it is known that the crystal structure of subdomain 2 of G-actin is slightly different between the ATP and ADP states26; besides, it was reported recently that the X-ray fiber diffraction of actin filaments showed that the angles between the subdomains 1, 2, and the subdomains 3, 4 in an actin monomer are slightly altered upon polymerization27. We calculated the p(r) functions using their reported structural data, but they were indistinguishable from those of ATP-G-actin (data not shown), implying that little structural alterations, such as small conformational changes in the subdomain orientation, are not clearly detectable in the p(r) analysis at the present resolution. Nevertheless, the data confirm the mono-dispersity of the solution within this resolution, implying that the G-actin solution at the initial stage of the following temperature-induced series of polymerization experiments was not contaminated with polymers.

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