title: 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-1actin in G-buffer). The SAXS experiments were started from the mono-disperse G-actin state, which was confirmed by comparing the scattering pattern inq- and real space with X-ray crystallographic data. We observed that the forward scattering intensityI(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 obtainedp(r) with the theoreticalp(r) for the helical and linear oligomers (2-13mers) calculated based on the X-ray crystallographic data. We found thatp(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.
authors.affiliation: Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Togo
authors.initials: T
authors.lastname: Shimozawa
authors.affiliation: Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Toshiko
authors.initials: T
authors.lastname: Fukasawa
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Ohtaki
authors.affiliation: Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Aramaki
authors.affiliation: Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Katsuzo
authors.initials: K
authors.lastname: Wakabayashi
authors.affiliation: Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan; Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan; Waseda Bioscience Research Institute in Singapore (WABIOS), 11 Biopolis Way, #05-01/02 Helios, 138667 Singapore.
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Ishiwata
keywords: X-ray scattering; actin trimer; oligomer distribution; solution structure