Enrique M. De La Cruz & Thomas D. Pollard
Dept. Cell Biology & Anatomy, Johns Hopkins Medical School, Baltimore, MD 21205 Salk Institute for Biological Studies, 10010 Torrey Pines Road, La Jolla, CA 92039
We have characterized the binding of rhodamine phalloidin to actin filaments from three highly divergent species (S. cerevisiae, Acanthamoeba and rabbit skeletal muscle) and muscle actin filaments saturated with either myosin subfragment-1 or tropomyosin to elucidate how minor differences in amino acid sequences and interaction with muscle regulatory proteins generate different biochemical and mechanical properties of actin filaments. Transient kinetic measurements of rhodamine phalloidin binding to rabbit muscle actin filaments indicate an association rate constant of 2.9 x 104 M-1 s-1 and a dissociation rate constant of 4.8 x 104 s-1. The ratio of the rate constants yields a dissociation equilibrium constant of 17 nM. When myosin subfragment-1 and tropomyosin are bound to actin filaments the rate constants for rhodamine phalloidin binding are the same as for actin alone demonstrating that these regulatory proteins do not affect the structure of actin in a way that disrupts the phalloidin binding site. Neither substitution of CaCl2 for MgCl2 nor the inclusion of 20 mM phosphate alters the rate or equilibrium constants. The association rate constant of rhodamine phalloidin is low for binding to an actin filament, suggesting that not all of the phalloidin binding sites on a filament are readily available but become accessible as the filament experiences thermal fluctuations in shape, or "breathes". We can eliminate other factors that slow the rates of binding such as slow protonation reactions and electrostatic interactions. Raising the solvent microviscosity, but not macroviscosity, inhibits filament breathing and phalloidin binding kinetics are inhibited. S. cerevisiae actin filaments bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant. A single amino acid substitution in the proposed phalloidin binding site of the yeast actin may explain its lower affinity for rhodamine phalloidin. Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more flexible and can breathe more easily than rabbit or Acanthamoeba actin filaments. The different mechanical properties of actin filaments may relate to the demands and functions of the actin cytoskeleton in each particular cell type.