Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay

Harada, Yoshie; Sakurada, Katsuhiko; Aoki, Toshiaki; Thomas, David D.; Yanagida, Toshio

doi:10.1016/s0022-2836(05)80060-9

Cited by 499 publications

(441 citation statements)

References 39 publications

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“…Actin was purified from rabbit back muscle as previously described (34). TRITC-labeled F-actin was produced by polymerizing G-actin in the presence of TRITC-phalloidin (35). Actin filaments decorated with full-length myosin molecules in the nucleotide-free rigor state were formed by adding 10 mM EDTA to a mixture of F-actin and myosin extract to chelate Mg 2ϩ of the myosin extraction buffer (modified from Ref.…”

Section: Methodsmentioning

confidence: 99%

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Amrute-Nayak

Antognozzi

Scholz

et al. 2008

Journal of Biological Chemistry

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In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times Muscle contraction involves the sliding of actin filaments relative to myosin filaments (1, 2), which is driven by cyclic interactions of the myosin head domains with the actin filaments, powered by hydrolysis of ATP. During each ATP-hydrolysis cycle chemical energy of ATP hydrolysis is transformed into mechanical work by a multistep power-stroke which drives the actin filaments several nanometers past the myosin filaments. The power-stroke is associated with the release of the ATP hydrolysis products, P i (inorganic phosphate) and ADP, from the active site (3). It is generally believed that P i release from the AM⅐ADP⅐P i 3 complex is closely related to the initiation of the power-stroke (4) and to the transition of the myosin head domain from states of weak and non-stereospecific actin binding to states of strong and stereospecific binding to actin (5-7). Because of the assumed close relation between power stroke and the release of inorganic phosphate from the myosin head domain, studying the effects of inorganic phosphate on contracting muscle fibers became a main element to elucidate the relation between P i release and force generation. Effects of P i were examined on isometric force (4, 8 -14) and unloaded shortening velocity (9, 13, 15) as well as on force transients in response to release of P i from caged P i (16,17). As a result of these studies it was proposed that inhibition of active force by inorganic phosphate results from rebinding of P i to the AM⅐ADP intermediate that is formed after P i release (cf. Scheme 1). Thus, the release of phosphate is reversed and a strong binding force generating cross-bridges in the AM⅐ADP state are reversed to a weak binding non-force generating AM⅐ADP⅐P i state (the AM⅐ADP⅐Pi I state in Scheme 1) that is in rapid equilibrium with the detached M⅐ADP⅐P i I intermediate. To fully account for the observed steady state kinetic data and the force transients recorded upon release of P i from caged P i , it was proposed that a strong binding AM⅐ADP⅐P i intermediate (the AM⅐AD...

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Section: Methodsmentioning

confidence: 99%

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Amrute-Nayak

Antognozzi

Scholz

et al. 2008

Journal of Biological Chemistry

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“…(The rate of ATP hydrolysis does not change greatly.) Under no load, the sliding distance per hydrolysis of ATP molecule in the steady state is probably longer than that in a single event (Harada et al 1990). The chemical energy consumption is well regulated, depending on the mechanical work required.…”

Section: The Loose Couplingmentioning

confidence: 99%

The loose coupling mechanism in molecular machines of living cells

Oosawa

2000

Genes to Cells

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Living cells have molecular machines for free energy conversion, for example, sliding machines in muscle and other cells,¯agellar motors in bacteria, and various ion pumps in cell membranes. They are constructed from protein molecules and work in the nm (nanometer), pN (piconewton) and ms (millisecond) ranges, without inertia. In 1980s, a question was raised of whether the input±output or in¯ux±ef¯ux coupling in these molecular machines is tight or loose, and an idea of loose coupling was proposed. Recently, the long-distance multistep sliding of a single myosin head on an actin ®lament, coupled with the hydrolysis of one ATP molecule, was observed by Yanagida's group using highly developed techniques of optical microscopy and micromanipulation. This gave direct evidence for the loose coupling between the chemical reaction and the mechanical event in the sliding machine. In this review, I will brie¯y describe a historical overview of the input±output problem in the molecular machines of living cells. Input±output in molecular machinesConsider a series of solid gears. The ratio of the angle of rotation of a gear in the input to the angle of rotation of another gear in the output is constant and independent of the speed of rotation or the torque for rotation. In such a way, are the input and output or in¯ux and ef¯ux in molecular machines of living cells tightly coupled? Does one chemical reaction in the input always generate a unit mechanical movement in the output?The molecular machines are very small, so that the input free energy is not much larger than the energy of thermal noise. Nevertheless, the machine is neither cooled nor isolated, but works at room temperature in water. The structural units of the machine, the protein molecules, are not rigid, but have thermal¯uctuation. The energy exchange among these units and their environment may be positively involved in the process of free energy conversion. The relation between input and output in the machine would not be straightforward. Intermediate processes in the machine would make the in¯ux±ef¯ux coupling loose. It is very likely that living cells invented the loose coupling mechanism in order to give an ef®cient conversion of small free energy in thermal noise.To understand the working principle of molecular machines, it is important to know, ®rst of all, their function. We have to de®ne their input and output, or in¯ux and ef¯ux, and carry out quantitative investigations on the relation between them under various conditions.

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“…Finally, we would like to point out that, although single molecule detection has been reported and opens up new possibilities with regard to mechanochemical coupling studies of motor proteins [4], the major discrepancies in the literature concern sliding over assemblies of myosin heads at low loads [6,7]. Thus, it may be desirable to follow the kinetics of nucleotide turnover by a few hundred adjacent myosin heads during unloaded sliding.…”

Section: Discussionmentioning

confidence: 99%

“…However, there is no direct evidence of how many such strokes may be derived from the hydrolysis of a single ATP molecule. Under conditions of low load where actin filaments are sliding near their maximum velocity, there remains a discrepancy in the estimation of the distance travelled per ATP molecule hydrolyzed as calculated from bulk assays [6,7].…”

Section: Introductionmentioning

confidence: 99%

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Conibear

Bagshaw

1996

FEBS Letters

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The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged-ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady-state hydrolysis of the ATP analog. These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.

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Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay

Cited by 499 publications

References 39 publications

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

The loose coupling mechanism in molecular machines of living cells

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

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