Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.

The products of MgATP hydrolysis bind to the nucleotide site of myosin and thus may be expected to inhibit the contraction of muscle fibers. We measured the effects of phosphate and MgADP on the isometric tensions and isotonic contraction velocities of glycerinated rabbit psoas muscle at 10 degrees C. Addition of phosphate decreased isometric force but did not affect the maximum velocity of shortening. To characterize the effects of ADP on fiber contractions, force-velocity curves were measured for fibers bathed in media containing various concentrations of MgATP (1.5-4 mM) and various concentrations of MgADP (1-4 mM). As the [MgADP]/[MgATP] ratio in the fiber increases, the maximum velocity achieved by the fiber decreases while the isometric tension increases. The inhibition of fiber velocities and the potentiation of fiber tension by MgADP is not altered by the presence of 12 mM phosphate. The concentration of both MgADP and MgATP within the fiber was calculated from the diffusion coefficient for nucleotides within the fiber, and the rate of MgADP production within the fiber. Using the calculated values for the nucleotide concentration inside the fiber, observed values of the maximum contraction velocity could be described, within experimental accuracy, by a model in which MgADP competed with MgATP and inhibited fiber velocity with an effective Ki of 0.2-0.3 mM. The average MgADP level generated by the fiber ATPase activity within the fiber was approximately 0.9 mM. In fatigued fibers MgADP and phosphate levels are known to be elevated, and tension and the maximum velocity of contraction are depressed. The results obtained here suggest that levels of MgADP in fatigued fibers play no role in these decreases in function, but the elevation of both phosphate and H+ is sufficient to account for much of the decrease in tension.

SUMMARY1. The effects of phosphate and protons on the mechanics and energetics of muscle contraction have been investigated using glycerinated rabbit psoas muscle.2. Fibres were fully activated by addition of Ca21 (pCa 4-5) at 10 'C. The velocities of contraction were measured in isotonic load clamps, and the velocities of unloaded fibres were measured by applying a series of step changes in fibre length. Fibre ATPase activity was monitored using an enzyme system to couple ADP production to reduced nicotinamide-adenine dinucleotide (NADH) and measuring the depletion of NADH by optical density.3. At pH 7-0 and 3 mM-phosphate, isometric tension (P0) was 13-2 +0-9 N/cm2 (mean+S.E.M., n = 10 observations), the maximum contraction velocity (Vmax) was 1-63 + 0-05 lengths/s (n = 5) and the ATPase activity was 1-27 + 0-12 s-' myosin head-' (n = 35). Increasing phosphate from 3 to 20 mm at pH 7 0 does not affect Vmax, causes a small decrease in the ATPase activity (15-20%) and decreases Po by approximately 20%. Changing pH from 7 to 6 at 3 mM-phosphate decreases Po by 45 % and both Vma. and ATPase activity by 25-30 %. The effects of changing both pH and phosphate were approximately additive for all parameters measured. The inhibition of these parameters by low pH and high concentration of phosphate was reversible.4. The force-velocity relation was fitted by the Hill equation using a non-linear least-squares method. The value of the parameter which describes the curvature, a/PO, was 0-20. The curvature of the force-velocity relation was not changed by addition of phosphate or by changes in pH.5. These data provide information on both the kinetics of the actomyosin interaction and on the process of muscle fatigue. The data are consistent with models of cross-bridge kinetics in which phosphate is released within the powerstroke in a step involving a rapid equilibrium between states. The inhibition by protons is more complex, and may involve less specific effects on protein structure.6. During moderate fatigue of living skeletal muscle, MgATP concentration is t To whom correspondence should be addressed.

We have explored a model of crossbridge kinetics that explains many of the effects on steady-state muscle contraction of ligands that bind to the nucleotide site on myosin. The mathematical model follows the basic framework for crossbridge function first established by A. F. Huxley. In the model, detached crossbridges initially bind in a weakly attached, A.M.D.Pi state (A, actin; M, myosin; D, ADP; Pi, orthophosphate) at the beginning of the region of positive force production. Pi release then results in transition to a strongly-bound A.M.D state, as has been suggested by other investigators from both biochemical and mechanical data. Mg2+ ADP release and subsequent crossbridge detachment due to Mg2+ ATP binding to the A.M state occur at the end of the region of positive force production. Work in a number of laboratories has now defined the effects on steady-state contraction of variations in the concentrations of Mg2+ ATP, Mg2+ ADP and Pi. These data provide valuable constraints that can be used to further refine current models. The maximum velocity of shortening (V max) and ATPase activity of muscle fibres exhibit classical saturation behaviour with respect to Mg2+ ATP concentration, with Mg2+ ADP acting as a competitive inhibitor. The model can reproduce this behaviour. The model also explains the observations that increasing [Mg2+ ATP] decreases isometric tension and increasing [Mg2+ ADP] increases tension. As the concentration of Pi increases, model predictions suggest that tension should decrease approximately as log[Pi], that ATPase activity should decrease less than tension and that V max should be almost unchanged, as has been found experimentally. The model also demonstrates that the connection between the parameters of contraction and the free energy of hydrolysis of Mg2+ ATP can be complex.