Sub-piconewton force fluctuations of actomyosin in vitro

Ishijima, Akihiko; Doi, Takashi; Sakurada, Katsuhiko; Yanagida, Toshio

doi:10.1038/352301a0

Cited by 252 publications

(140 citation statements)

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“…1b). The size of the displacements caused by kinesin were determined accurately from the bead displacements by correcting for the effect of kinesin-to-bead stiffness using a previously described method (13,26) with modifications ( Fig. 1 b and c).…”

Section: Resultsmentioning

confidence: 99%

“…Detection of the single-channel current was much easier than that of force and displacement by single-motor protein molecules because a huge number of ions (about 10 6 ) flow during single-channel openings and energy consumed by the ion flow is about 10 5 -fold greater than the free energy (10 Ϫ19 J) liberated by hydrolysis of single ATP molecules that promote the movement of motor proteins. Recent progress in the measurement of force and displacement of single kinesin or myosin molecules provides the opportunity to directly determine their mechanical properties (10)(11)(12)(13)(14)(15). The resolution of the measurement system is about 10 Ϫ20 J, which is close to the energy consumed by the flow of single ions through a channel.…”

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confidence: 99%

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Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

Higuchi

Muto

Inoue

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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121

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To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesinmicrotubule rigor complex using the optical trap, and ATP was released by f lash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 M ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 M ؊1 ⅐s ؊1 , followed by force generation at 45 s ؊1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.Motor proteins, such as kinesin, myosin, dynein, and RNA polymerase, convert the chemical energy of ATP hydrolysis into mechanical work. To investigate the mechanism of energy transduction of motor proteins, the elementary mechanical steps like force generation and sliding should be related to chemical reactions such as ATP binding, and P i and ADP release. The biochemistry of ATP hydrolysis by kinesin in solution has been investigated using native and recombinant proteins, with or without microtubules. The ATPase rate of kinesin is very low (Ͻ0.01 s Ϫ1 ) in the absence of microtubules at room temperature and is activated more than 2,000-fold by microtubules to Ͼ20 s Ϫ1 (1-5). The intermediate states of the kinesin-microtubule complex are similar to those of actomyosin, that is, M⅐K, M⅐K⅐ATP, M⅐K⅐ADP⅐P i , and M⅐K⅐ADP, where M, K, and P i are microtubule, kinesin, and inorganic phosphate, respectively. The rate-limiting step of the ATPase cycle is ADP release in the absence of microtubules, and P i or ADP release in the presence of microtubules (1-5).These biochemical results cannot be correlated directly to the mechanical cycle of kinesin, because the movements are not detected in solution. A powerful method for the investigation of mechanochemical coupling is to detect the mechanical change after laser photolysis of caged compounds, e.g., caged ATP, caged ADP, and caged P i (6, 7). Caged compounds photolyze within several milliseconds after a pulse of ultraviolet light. The mechanical reactions of actomyosin in muscle have been detected after release of ATP by photolysis of caged ATP. The rate of actomyosin dissociation after ATP release and rate of force generation were determined (6).In the experimental systems containing many molecules, individual events are averaged and the state of each molecule is not determined. Analysis of the events produced by single molecules reveals the molecular functions more directly. The kinetics of single ion channels were elucidated by stochastic analys...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

Higuchi

Muto

Inoue

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

121

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“…The sliding force was measured by attaching the actin ®lament to the tip of a very thin glass needle (Kishino & Yanagida 1988). The force required to stop the sliding was a few pN per myosin head, which was also of the same order as was expected from muscle ®bres (Ishijima et al 1991). Thus, a sliding machine was composed of single actin ®laments and single myosin heads ®xed on a plate.…”

Section: The Unit Machine Of Slidingmentioning

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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“…In the presence of fuel, which in the cell ist adenosinetriphosphate (ATP), the motor starts moving in a direction defined by the polarity of the track filament. Experimental methods to study the physical properties of motor proteins have been developed in recent years [3][4][5][6][7].…”

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confidence: 99%

Spontaneous Oscillations of Collective Molecular Motors

1997

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We analyze a simple stochastic model to describe motor molecules which cooperate in large groups and present a physical mechanism which can lead to oscillatory motion if the motors are elastically coupled to their environment.Beyond a critical fuel concentration, the non-moving state of the system becomes unstable with respect to a mode with angular frequency ω. We present a perturbative description of the system near the instability and demonstrate that oscillation frequencies are determined by the typical timescales of the motors.

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Sub-piconewton force fluctuations of actomyosin in vitro

Cited by 252 publications

References 43 publications

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

The loose coupling mechanism in molecular machines of living cells

Spontaneous Oscillations of Collective Molecular Motors

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