Size distribution of linear and helical polymers in actin solution analyzed by photon counting histogram

¹ Waseda University
² University of Tokyo

^* To whom correspondence should be addressed. E-mail: funatsu{at}mail.ecc.u-tokyo.ac.jp.

Submitted on October 5, 2006
Revised on November 6, 2006
Accepted on 22 November 2006

	Abstract

Actin is a ubiquitous protein, which is a major component of the cytoskeleton playing an important role in muscle contraction and cell motility. At steady state actin monomers and filaments (F-actin) coexist, and actin subunits continuously attach and detach at the filament ends. However, the size distribution of actin oligomers in F-actin solution has never been clarified. Here we investigated the size distribution of actin oligomers using the photon counting histograms. For this purpose, actin was labeled with a fluorescent dye, and the emitted photons were detected by the confocal optics (the detection volume was of femtoliter (fL) order). Photon counting histograms were analyzed to obtain number distribution of actin oligomers in the detection area from the brightness of actin oligomers, assuming that the brightness of an oligomer was proportional to the number of protomers. We found that the major populations at physiological ionic strength were 1-5mers. For data analysis we successfully applied the theory of linear and helical aggregations of macromolecules(1). The model postulates the three states of actin, i.e., monomers, linear polymers, and helical polymers. Here we obtained three parameters: the equilibrium constant for the polymerization of linear polymers K_l = (5.2 ± 1.1)x10⁶ M^-1, that of helical polymers K_h = (1.6 ± 0.5)x10⁷ M^-1, and the ratio of helical to linear trimers = (3.6 ± 2.3)x10^-2. The excess free energy of transforming a linear trimer to a helical trimer, which is assumed to be a nucleus for helical polymers, was calculated to be 2.0 kcal/mol. These analyses demonstrate that the oligomeric phase at steady state is predominantly composed of linear 1-5mers, and the transition from linear to helical polymers occurs on the level of 5-7mers.

Key Words: critical concentration for polymerization of actin, linear polymer of actin