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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.

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Related in: MedlinePlus

Effect of temperature on polymerization of actin with different divalent cations and nucleotides.Relative average molecular weight of actin aggregates in solution, M̄w(T)/Mw(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.
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f3-6_1: Effect of temperature on polymerization of actin with different divalent cations and nucleotides.Relative average molecular weight of actin aggregates in solution, M̄w(T)/Mw(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.

Mentions: As shown in Fig. 3, the formation of actin oligomers upon increasing temperature is evident from M̄w(T)/Mw(T0). The circles and the squares in Fig. 3 indicate the non-fibrous and fibrous nature of actin polymers, respectively, in terms of the absence and presence of the fiber diffraction pattern in I(q). We call the temperature at which the emergence of the fiber diffraction peak, a measure of the formation of F-actin, becomes apparent, a characteristic temperature, TF. We anticipate that above TF the actual length of the actin polymers, Dmax, certainly exceeds the maximum resolution of our SAXS experiments, π/qmin∼40 nm, where qmin is the accessible minimum scattering vector23. Simultaneously, the evaluation of I(q→0,T) inevitability becomes less reliable, thereby giving perhaps underestimated average molecular weights.


Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering
Effect of temperature on polymerization of actin with different divalent cations and nucleotides.Relative average molecular weight of actin aggregates in solution, M̄w(T)/Mw(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.
© Copyright Policy
Related In: Results  -  Collection

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

f3-6_1: Effect of temperature on polymerization of actin with different divalent cations and nucleotides.Relative average molecular weight of actin aggregates in solution, M̄w(T)/Mw(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.
Mentions: As shown in Fig. 3, the formation of actin oligomers upon increasing temperature is evident from M̄w(T)/Mw(T0). The circles and the squares in Fig. 3 indicate the non-fibrous and fibrous nature of actin polymers, respectively, in terms of the absence and presence of the fiber diffraction pattern in I(q). We call the temperature at which the emergence of the fiber diffraction peak, a measure of the formation of F-actin, becomes apparent, a characteristic temperature, TF. We anticipate that above TF the actual length of the actin polymers, Dmax, certainly exceeds the maximum resolution of our SAXS experiments, π/qmin∼40 nm, where qmin is the accessible minimum scattering vector23. Simultaneously, the evaluation of I(q→0,T) inevitability becomes less reliable, thereby giving perhaps underestimated average molecular weights.

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