The experimental setup of the dual-beam optical trap is described in detail elsewhere (49). All experiments were performed at 23°C. Dead heads of purified myosins were eliminated as described above. The eGFP tag at the C-terminal end of the myosin was used for surface attachment through binding with anti-GFP antibody. The nitrocellulose-coated glass surface of the sample chamber was also coated with 1.5-μm-diameter silica beads that acted as platforms. The steps followed for preparing the chamber were as follows: 20 μl of anti-GFP antibody (~0.01 mg/ml; Abcam) was flowed through the chamber; 20 μl of AB buffer containing BSA (1 mg/ml) (ABBSA buffer) was flowed through to block the exposed surface; the surface was sparsely coated with myosin by flowing through ~50 to 200 pM of human β-cardiac sS1; the chamber was washed with 20 μl of AB buffer; 20 μl of ABBSA buffer containing 250 to 500 pM ATP, TMR-phalloidin–labeled biotin-actin filaments, neutravidin-coated polystyrene beads (Polysciences), and the oxygen-scavenging and ATP regeneration systems described above was flowed through the chamber; and the chamber was sealed with vacuum grease to stop evaporation of the solution. Two neutravidin-coated polystyrene beads (1 μm diameter) were trapped in two different laser beams. Bead-actin-bead assembly, also known as a dumbbell, was formed using the trapped beads bound to each end of a TMR-phalloidin– and biotin-labeled actin filament. The dumbbell was stretched to remove compliance in the actin filament and brought close to the bead pedestal on the surface for interaction with myosin. A trap stiffness of ~0.1 pN/nm was used.

The data collected from individual molecules were analyzed for force measurements, and such experiments were performed using myosins from at least two protein purifications. The number of total force events from individual molecules on average was 20 to 300. We have always observed that the force distribution of all myosin constructs is accompanied with a long tail (fig. S1), as reported previously (49). Each force distribution was fitted to a double-Gaussian function to take account of the smaller population of higher force events in the analysis (fig. S1). The major peak of the fit yielded the intrinsic force of an individual molecule reported here. Such intrinsic force values of multiple molecules were used to calculate the mean force value (Fig. 1B).

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