Thermogenesis by resting muscle varies with conditions and plays an active role in homeostasis of body weight. The low metabolic rate of living resting muscles requires that ATP turnover by myosin be inhibited relative to the purified protein in vitro. This inhibition has not been previously seen in in vitro systems. We used quantitative epifluorescence microscopy of fluorescent nucleotides to measure single nucleotide turnovers in relaxed, permeable skeletal muscle fibers. We observed two lifetimes for nucleotide release by myosin: a fast component with a lifetime of approximately 20 s, similar to that of purified myosin, and a slower component with a lifetime of 230 +/- 24 s. We define the latter component to be the "super relaxed state." The fraction of myosins in the super relaxed state was decreased at lower temperatures, by substituting GTP for ATP or by increased levels of myosin phosphorylation. All of these conditions have also been shown to cause increased disorder in the structure of the thick filament. We propose a model in which the structure of the thick filament modulates the nucleotide turnover rates of myosin in relaxed fibers. Modulation of the relative populations of the super relaxed and conventional relaxed states could have a profound effect on muscle thermogenesis, with the capacity to also significantly alter whole-body metabolic rate.

We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron paramagnetic resonance spectroscopy reveals that the pocket is in equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin, the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH0 and ΔS0, even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work-generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket and the observation that fast myosins are less efficient than slow myosins.

The mechanisms responsible for the inhibition of shortening velocity that occurs during muscle fatigue have not been completely elucidated. Phosphorylation of the myosin regulatory light chain (RLC) occurs during heavy use; however, previous reports on its role in affecting velocity have been equivocal. To further understand the process of fatigue, we varied the levels of myosin RLC phosphorylation (from 10 to >50%) and the concentrations of protons (from pH 7 to 6.2) and phosphate (from 5 to 30 mM), all of which change during fatigue. We measured the mechanics of permeable rabbit psoas fibers at a temperature closer to physiological (30 degrees C), using a temperature jump protocol to briefly activate the fibers at the higher temperature to preserve sarcomere homogeneity. Although lowered pH alone had an effect on velocity, it was the three factors together, i.e., high phosphorylation, low pH, and high phosphate, that acted synergistically to inhibit fiber velocity by approximately 40%. Our data demonstrate that in conditions that simulate physiological muscle fatigue, myosin phosphorylation does contribute to the inhibition of contraction velocity of fully activated fast muscle fibers.

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.

The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1-2 mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P'), the absolute value of P' being nearly constant, approximately 0.04 N/mm(2), in variable ATP levels, pH 7. Tension decreased (50-60%) at pH 6, but upon addition of SL-ADP, P' was still approximately 0.04 N/mm(2). The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K(i), of approximately 240 and 280 microM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.

The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, ϵADP binds aggregated S1 with reduced affinity, and the bound ϵADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by p-phenylenedimaleimide is less efficient for aggregated S1·MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism.

A series of ATP analogs, in which moieties of various sizes have been added to the gamma-phosphorus of ATP, bind to the active site of myosin and to the actomyosin complex in myofibrils and in chemically skinned fibers. The affinity of the analogs for the active site shows only a slight dependence on the size of the added moiety. Addition of even our smallest group (CH3) reduced the binding affinity of ATPgamma-CH3 for S1 to 40 microM, a factor of 10(5) less than observed for ATP. Computer molecular docking of ATP-gammaCH3 into the myosin-ADP.BeF3 crystal structure of Dictyostelium discoideum indicates no steric interference to prevent binding. This suggests that the maintenance of charge at the gamma-phosphate is crucial for tight nucleotide binding. Addition of larger groups, (1) an EPR probe (ATP-gammaSL) or (2) ADP (i.e., P1, P5-diadenosine pentaphosphate, AP5A), reduced the affinity by only approximately a factor of 10 over that of ATP-gammaCH3. In the crystal structure of S1 complexed with nucleotides, the phosphates are buried within a protein structure called "the phosphate tube". Both the bulk of the modifying groups and the lack of dependence on the size of the group are incompatible with threading of the phosphates down the Pi-tube, showing that the tube must open. Similar domain movements have been found in other proteins including members of the G-protein superfamily, a family that has structural homologies to myosin.

1. Inhibition of actomyosin function by decreased pH has been proposed to account for much of the depression of muscle function during fatigue. The clearest support for this hypothesis has been from studies of skinned skeletal muscle fibre mechanics at low temperatures (< or = 15 degrees C). 2. We re-examined the effect of decreased pH (7.0-6.2) on skinned mammalian skeletal fibre mechanics at low (10 degrees C) and high (30 degrees C) temperatures, using recently developed protocols that allow reproducible mechanical data to be obtained at higher temperatures. 3. At 10 degrees C we duplicated previous observations of a significant inhibition of maximum shortening velocity (Vmax) and isometric tension (Po) by acidosis. In contrast, at the higher temperature, we found only a very minimal effect of acidosis on Vmax and a threefold reduction in the decrease in Po. 4. Thus at temperatures only slightly below physiological for mammalian skeletal muscle systems, pH plays a much less important role in the process of muscle fatigue at the cross-bridge level than has been suggested by data obtained at physiologically unrealistic temperatures.