Elastic distortion is fundamental in the generation of contractile forces by myosins. Resistance to elastic distortion (stiffness) of a cross-bridge determines the contribution to active force when a myosin head performs its power stroke under isometric conditions. With hypertrophic cardiomyopathy-related mutations we showed that elastic distortion takes place in and near the converter domain. From structural rearrangements around the converter, seen by protein crystallography for different states of the myosin head, we hypothesized that stiffness of the myosin head changes during its power stroke.

We tested this hypothesis with myosin Va as this myosin pauses sufficiently long before executing the second part of its power stroke. Thus, we could determine stiffness of an individual myosin head by three-bead optical trapping before and after the second part of the power stroke, denoted by a sub-step in the binding events.

We determined stiffness of the myosin head by ramp-shaped stage movements. Upon binding of the myosin head to the actin filament the dumbbell becomes displaced but with an amplitude smaller than that of stage movement, due to increasing elastic extension of the head domain. With the sub-step, however, dumbbell movement suddenly followed the imposed stage movement more closely than before, indicating a suddenly reduced elastic extension of the myosin head. Quantitative analysis revealed a 2- to 3-fold increase in myosin head stiffness with the sub-step.

We conclude that stiffness of myosin increases substantially with reorientation of its lever arm towards the post-power stroke conformation in the second part of the power stroke.