Myosin lever disposition during length oscillations when power stroke tilting is reduced

Abstract

(I M3) from tetanized, intact skeletal muscle fiber bundles was measured during sinusoidal length oscillations at 2.8 kHz, a frequency at which the myosin motor's power stroke is greatly reduced. I M3 signals were approximately sinusoidal, but showed a "double peak" distortion previously observed only at lower oscillation frequencies. A tilting lever arm model simulated this distortion, where I M3 was calculated from the molecular structure of myosin subfragment 1 (S1). Simulations showed an isometric lever arm disposition close to normal to the filament axis at isometric tension, similar to that found using lower oscillation frequencies, where the power stroke contributes more toward total S1 movement. Inclusion of a second detached S1 in each actin-bound myosin dimer increased simulated I M3 signal amplitude and improved agreement with the experimental data. The best agreement was obtained when detached heads have a fixed orientation, insensitive to length changes, and similar to that of attached heads at tetanus plateau. This configuration also accounts for the variations in relative intensity of the two main peaks of the M3 reflection substructure after a length change. This evidence of an I M3 signal distortion when power stroke tilting is suppressed, provided that a large enough amplitude of length oscillation is used, is consistent with the tilting lever arm model of the power stroke. skeletal muscle; X-ray diffraction; muscle mechanics; molecular motors; subfragment 1 structure MYOSIN MOTORS are intracellular protein macromolecules, which function as actin-dependent transducers of ATP hydrolysis free energy into mechanical work. At least 17 classes of myosin exist, fulfilling many important motile roles in cell physiology, including cell division, cytokinesis, exo-, endo-, and pinocytosis, acuity adjustment of sensory receptors, and the contraction of all forms of muscle tissue (34). Their mechanism is thought to involve the active tilting (power stroke) of the myosin lever arm domain [a 9-nm-long projection from the actin-and ATP-binding motor domain of myosin subfragment 1 (S1)] during ATP cleavage at the motor domain's catalytic site (17, 31). Tilting displaces the arm's tip, producing a ϳ4-to 10-nm translational movement or 2-4 pN of force (7,27). A feature unique among myosin motors is that striated muscle myosin (myosin II) aggregates into highly structured filaments aligned within a quasicrystalline lattice, which yield a characteristic X-ray diffraction pattern (18) and the intensity of the 14.55 nm meridional X-ray reflection (I M3 ) is thought to be sensitive to lever arm tilting (21). This is because I M3 depends on the density of the S1 mass projected onto the filament axis, which changes with lever arm tilting. The maximum I M3 (I M3,max ) is reached when the lever is roughly perpendicular to the filament because at this point the axially projected mass distribution is at its most concentrated. In isometrically activated muscle, I M3 increases to a maximum for a small filament displacement ...

Effects of solution tonicity on crossbridge properties and myosin lever arm disposition in intact frog muscle fibres

“…6, that I M3 calculated with the model tends to underestimate the double peak distortion compared with the experimental results. The reasons for this deviation have been discussed previously (Griffiths et al 2005). One possible reason is that the tilting mass is greater than the lever arm only considered in the model.…”

“…7) suggesting a shift in the mean lever arm position towards the position of maximum intensity (towards the Z‐line). The fitting of the X‐ray data with the model calculation (Griffiths et al 2005) showed that in five experiments at 12°C, 1.4T saline shifted the mean lever arm position (Δ y ) from 0.77 ± 0.17 nm in isotonic saline to 1.70 ± 0.31 nm in hypertonic solution, consistent with a shift in lever arm tilt to displace the lever arm tip away from the Z‐line by 0.93 nm. This is the direction of lever arm displacement we observed in other studies where the force per crossbridge was reduced by other means (Griffiths et al 2002).…”

“…The mechanism of the double peak distortion has been reported previously (Griffiths et al 2002), briefly the distortion occurs because the increase in I M3 during the release phase of the sinusoid, is reversed if the lever arm moves beyond its position at maximum intensity (lever arm almost perpendicular to filament axis). The presence of this distortion allows calculation of the mean lever arm position by using a model which simulates the I M3 changes expected from the oscillatory tilting of the lever arm induced by the bundle length oscillations, using the crystallographic structure of myosin (Griffiths et al 2005). The model analysis showed that the reduction of the double peak distortion in hypertonic solution can be accounted for by a change in the mean lever arm tip position of 0.93 nm away from the position of maximum intensity (away from the Z‐line).…”

“…Meridional X‐ray patterns were analysed by a curve fitting procedure (Bagni et al 2001) to obtain integrated intensity over the whole reflection. The variation in intensity during a single length oscillation was then fitted to the theoretical intensity variation obtained from the displacement of the lever arm of a model of the double‐headed myosin dimer, one head being free, the other actin‐bound (Griffiths et al 2005), for the imposed length changes. This lever arm displacement was calculated as half the sarcomere length change minus the extension of series elasticity (taken as one half of total compliance), which may be estimated from the force changes during the oscillations.…”

Effects of the Number of Actin-Bound S1 and Axial Force on X-Ray Patterns of Intact Skeletal Muscle

“…An ordered, free S1 was assumed to be associated with each actin-bound S1 to obtain the highest I M3 sensitivity to length changes (20) and in agreement with studies of the effects of x-ray scattering from opposite halves of the thick filament (21), which are best explained with the inclusion of such a free S1 component. A more detailed description of the simulation procedure is given elsewhere (16,20,22).…”