Skeletal muscle shortens faster against a lower load. This force-velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre-microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere.
We study the kinetics of the overstretching transition in λ-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25°C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5-2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of ~25 basepairs. This mechanism increases the free energy for the elementary reaction to ~94 k(B)T, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.
Key pointsr Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin-actin interactions increases with the increase in shortening velocity.r We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration.r A model simulation of the myosin-actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation.r These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction. AbstractThe chemo-mechanical cycle of the myosin II-actin reaction in situ has been investigated in Ca 2+ -activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (P i ) concentration. It was found that in control conditions (no added P i ), shortening at V ࣘ 350 nm s -1 per half-sarcomere, corresponding to force (T) greater than half the isometric force (T 0 ), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T 0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising P i concentration by 10 mM, which reduces T 0 and the number of motors by 40-50%, does not influence the dependence on V of number and strain. A model simulation of the myosin-actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that P i and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction. Abbreviations A, actin monomer to which a myosin motor attaches; A , actin monomer next to A, 5.5 nm away from the centre of the sarcomere; CD, catalytic domain; CSA, fibre cross-sectional area; hs, half-sarcomere; LCD, light chain domain; M, myosin motor; f, fraction of actin-attached motors relative to the number attached in isometric contraction in control conditions; P i , inorganic phosphate.
The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (Cf) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa−1. This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa−1. The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm−1) is smaller than that of frog (2.78 ± 0.30 pN nm−1). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog.
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