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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

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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.


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 model24. The structure of a linear polymer was produced from the helical structure (see text).
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f4-6_1: 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 model24. The structure of a linear polymer was produced from the helical structure (see text).

Mentions: For a quantitative analysis, we attempted to compare the p(r) functions in solution obtained by SAXS with the theoretical distributions calculated from the crystal structures of actin filaments. The oligomer models were constructed from the F-actin structure, which was derived by fitting the atomic structure of G-actin determined from the X-ray crystallography to the X-ray fiber diffraction data from the oriented sols of F-actin24. Using the PDB coordinates (ftp://149.217.48.3/pub/holmes/pdb/crystal_structure_actin_helix.pdb), we calculated p(r) functions for helical polymers (H) ranging from a dimer (H2) up to a 13mer (H13) (Fig. 4a). We also calculated the p(r)s for the linear actin polymers (L) ranging from a dimer (L2) to a 7mer (L7) (Fig. 4b). Since the linear form has not been established yet, the linear polymers were created by simply removing one of the two major helical strands as shown in Fig. 4c.


Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering
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 model24. The structure of a linear polymer was produced from the helical structure (see text).
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Related In: Results  -  Collection

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f4-6_1: 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 model24. The structure of a linear polymer was produced from the helical structure (see text).
Mentions: For a quantitative analysis, we attempted to compare the p(r) functions in solution obtained by SAXS with the theoretical distributions calculated from the crystal structures of actin filaments. The oligomer models were constructed from the F-actin structure, which was derived by fitting the atomic structure of G-actin determined from the X-ray crystallography to the X-ray fiber diffraction data from the oriented sols of F-actin24. Using the PDB coordinates (ftp://149.217.48.3/pub/holmes/pdb/crystal_structure_actin_helix.pdb), we calculated p(r) functions for helical polymers (H) ranging from a dimer (H2) up to a 13mer (H13) (Fig. 4a). We also calculated the p(r)s for the linear actin polymers (L) ranging from a dimer (L2) to a 7mer (L7) (Fig. 4b). Since the linear form has not been established yet, the linear polymers were created by simply removing one of the two major helical strands as shown in Fig. 4c.

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.