Mechanical properties of single protein motor of muscle studied by optical tweezers.

Using optical tweezers and a dual-view (fluorescence and phase-contrast) microscope system, we have succeeded in (1) determining the location of heavy-meromyosin (HMM) molecules adhering to a glass surface precoated with nitrocellulose; and (2) measuring the binding force between an HMM molecule and an actin filament in the absence of ATP, i.e., the tensile strength that is the force needed to break the rigor bond. A polystyrene bead (1 ,um in diameter), selectively attached to the rear end of an actin filament through an actin binding protein, gelsolin, was captured by a single light beam of a YAG-laser (wavelength, 1.064 ,um). The optical gradient trap force was proportional to the displacement of the bead out to 200 nm. The actin filament (1-10 ,um in length) labeled with the fluorescent probe rhodamine phalloidin was observed under a fluorescence microscope using a charge-coupled device camera equipped with an image intensifier. Fig. 1 shows the single actin filament that was bound to the glass surface precoated with 1 ,ug/ml HMM. When the bead (arrow in Fig. 1 A) was trapped and pulled to the right at a constant rate, a part of the filament was stretched straight from the nodal point (arrowhead in Fig. 1 B). As the load imposed by optical tweezers was increased, the nodal point was broken and the actin filament was loosened (Fig. 1 C). As the bead was pulled further, the actin filament became taut at another nodal point (arrowhead in Fig. 1 D), which broke after a while. Thus, we could directly count the number ofHMM molecules that had attached to the filament. We previously reported the stepwise motion of an actin filament under low concentrations of ATP on the surface precoated with 1 jig/ml HMM (Miyata et al., 1994). Using the technique described here, the average number of HMM molecules that had attached to 1 ,um actin filament was estimated to be about 1.5, indicating that the stepwise motion was produced by one to several HMM molecules. We measured the tensile strength and the elasticity of an acto-HMM complex by analyzing the position of the bead with the spatial resolution of a nanometer scale under a phase-contrast microscope. When the trap center was moved at 100 nm/s, the average value of the tensile strength was 6.5 ± 3.7 pN (n = 6). The elasticity, estimated from a steep portion of a stress-strain relation obtained before breaking the rigor bond, was 0.3 ± 0.2 pN/nm (n = 3). This value is similar to the elasticity of the crossbridge estimated in the muscle fibers (Tawada and Kimura, 1986). On the other hand, the maximum strain (-70 nm) was large compared with the size of the myosin head (20 nm), so that the strain might contain the elongation of the elastic parts in addition to the myosin head. At present, we are conducting the same type of measurements on a silanized glass surface because nitrocellulose might contribute to elasticity. In addition, because a single rigor bond must be broken stochastically, we have started to measure the time required for breaking the rigor bond under a constant load. Those results will be published in the near future. A