Myosin molecular motors transduce free energy of ATP hydrolysis into work. Their proposed mechanism is rotation of the myosin S1 subfragment tail domain during S1 binding to actin. Proof of this rotation in a working myosin motor is crucial to acceptance of this mechanism. Uniquely, muscle myosin (myosin II) exists as highly structured filament aggregates of myosin dimers, from which three ‘crowns’ of S1 pairs project at axial intervals of 14.32 nm helically along the filament. In skeletal muscle, these filaments are parallel and radially aligned, giving rise to a quasi-crystalline structure which diffracts X-rays to produce a characteristic pattern. Time resolution of the muscle pattern intensity has increased dramatically in the past 40 years, from hours to microseconds, allowing use of X-ray diffraction as a time-resolved probe of S1 action.

The meridional reflection at 14.32 nm undergoes large intensity changes dependent on the occupancy of the ATP binding site and on the actin-bound S1 load. Its intensity (I_M3) falls upon rapid stretch or release of a muscle. This fall is thought to result from an elastic tail domain angular displacement and a subsequent active, ‘power stroke’ tail rotation. However, in the case of a release, I_M3 first passes through an intensity maximum (I_M3,max), occurring when tail disposition produces the most concentrated S1 axial mass projection (Piazzesi et al. 1995).

We applied sinusoidal length changes (amplitude 5 nm per half sarcomere p-p, 100Ð3000 Hz, 4 °C) to isolated muscle fibres (tibialis anterior from decapitated Rana temporaria) to examine the relation between I_M3 and fibre length. At frequencies ²ge³ 1 kHz, I_M3 signals were sinusoidal, in phase with sarcomere length with peak intensity at maximum shortening. As frequency was reduced below 1 kHz, I_M3 signals became increasingly distorted, forming a new intensity minimum at maximum shortening, causing a double I_M3 peak about this point. Simulation of both tail domain displacement and I_M3 showed that this behaviour was explicable as summation of elastic and active tail rotations: at ²ge³ 1 kHz, oscillations were fast compared with the power stroke, and I_M3 arose chiefly from elastic motion of the tail; below 1 kHz, active tail displacement proceeded further, increasing total angular tail displacement and carrying it through its position at I_M3,max during shortening, thus forming a new intensity minimum at maximum shortening. At all frequencies, we found an axial shortening of < 0.9 nm per half sarcomere was required to displace the tail domain to its I_M3,max position in our simulations (Bagni et al. 2001). Mean force during oscillations was ²le³ 5 % of isometric tension at all frequencies. Because distortion was absent at high frequencies, we could not determine tail disposition in this range, nor could we reject the possibility that the I_M3 double peak depended on the presence of a power stroke component rather than on axial displacement. To test this, we used shorter fibres in which the applied oscillations caused a proportionally greater change in sarcomere length. We found a similar I_M3 distortion to that at lower frequencies, in spite of a severely truncated active I_M3 component (33 % of tail motion at 200 Hz; 8 % at 3 kHz; mean position 0.92 nm from I_M3,max).

To test our method, we used the finding that temperature elevation increases S1 force. According to the power stroke theory, this should displace the tail domain towards I_M3,max. As predicted, we found I_M3 distortion increased as temperature rose, consistent with a 0.73 nm shift in tail mean position towards I_M3,max for a 28 % increase in tension in our simulations. Simulations also showed that this effect was not accounted for by an increased power stroke contribution to I_M3 at high temperature, or by changes in power stroke kinetics.

We conclude that, by use of the displacement required to reach an intensity reference point provided by I_M3,max, we have a complementary and independent method of determination of tail domain disposition to the Fourier synthesis determination of S1 structure by muscle fibre X-ray diffraction.

This work was supported by EU Transnational Access contract HPRI-CT-1999-00033.

All procedures accord with current local guidelines.