A single myosin head moves along an actin filament with regular steps of 5.3 nanometres

Kitamura, Kazuhiro; Tokunaga, Makio; Iwane, Atsuko H.; Yanagida, Toshio

doi:10.1038/16403

Cited by 529 publications

(384 citation statements)

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“…Using this technique, in combination with new manipulation techniques, a minimum unit of the sliding machine was constructed and simultaneous measurements of sliding and ATP hydrolysis were performed (Ishijima et al 1998;Kitamura et al 1999). A myosin head attached to an extremely thin needle was found to slide on several actin molecules along the actin ®la-ment, during or after the hydrolysis of one ATP molecule (Fig.…”

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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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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“…Because of the presence of series compliance in the thin filament and in other sarcomeric structures (Oosawa et al 1972;Huxley et al 1994;Wakabayashi et al 1994;Higuchi et al 1995), ~0.6 nm of the length change is applied at the cross-bridge level. This value is very much smaller than the step size (5.3 nm; Kitamura et al 1999), hence there is a possibility that elementary steps of the cross-bridge cycle can be studied.…”

Section: Methods Of Studying Cross-bridge Kineticsmentioning

confidence: 96%

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Kawai

Ishiwata

2006

J Muscle Res Cell Motil

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The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca 2+ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation. KeywordsActin; Regulatory proteins; Tropomyosin; Troponin; Myosin; Gelsolin; Cross-bridge kinetics; Sinusoidal analysis; BDM Thin filament reconstituted muscle fibre systemFor the purpose of understanding the cross-bridge interaction with actin and the role of regulatory proteins in this interaction, we reconstituted the thin filament in cardiac muscle fibres. This method is schematically represented in Fig. 1. The thin filament is first removed by gelsolin (Fig. 1A to 1B), followed by sequential reconstitution with G-actin (Fig. 1C), and regulatory proteins, tropomyosin (Tm) and troponin (Tn) (Fig. 1D). Gelsolin is an actin and thin filament severing protein, hence its action is specific and it does not disrupt other sarcomeric structure. However, overtreatment with gelsolin would disrupt the Z-line structure, because actin is one of the main constituents in the Z-line. The thin filament reconstitution method was initially developed at Ishiwata's laboratory and the key properties of the Correspondence to: Masataka Kawai, masataka-kawai@uiowa.edu. reconstituted fibres, such as the recovery of isometric tension, the tension-pCa relationship, and the function of spontaneous oscillatory contraction, were examined on skeletal fibres (Funatsu et al. 1994), followed by cardiac fibres (Fujita et al. 1996; Ishiwata 1998, 1999; for brief review see Ishiwata et al. 1998). In these works, it was demonstrated that bovine cardiac fibres were successful as the reconstituted system, in which actin, Tm and Tn can be replaced with those prepared from other muscles, for example, rabbit skeletal muscle proteins (Fujita et al. 1996;Fujita and Ishiwata 1999). The reconstitution method in cardiac fibres was successfully applied at ...

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“…[4][5][6][7] Several studies also probed the behavior of mechanoenzymes such as myosin, kinesin and dynein; most prominent were those techniques that measured the step size and force exerted by these proteins as they travel along actin filaments or microtubules. [8][9][10][11][12][13][14][15][16][17][18][19] Early work in single-molecule bioscience also included measurements of individual transcribing molecules of bacterial RNA polymerase, [20][21][22] and more recent studies with this enzyme were carried out to greater precision with instrumentation capable of making real time measurements with spatial resolution approaching one base pair. [23][24][25][26] Single-molecule methods have also been used to examine other nucleic acid motors like bacteriophage lambda exonuclease, 27,28 DNA polymerases, [29][30][31][32] Rad54 and Rdh54 DNA translocases, [33][34][35] the chromatin remodeling complexes RSC and Swi/Snf, 36,37 and several different helicases.…”

Section: Introductionmentioning

confidence: 99%

The importance of surfaces in single-molecule bioscience

2008

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The last ten years have witnessed an explosion of new techniques that can be used to probe the dynamic behavior of individual biological molecules, leading to discoveries that would not have been possible with more traditional biochemical methods. A common feature among these singlemolecule approaches is the need for the biological molecules to be anchored to a solid support surface. This must be done under conditions that minimize nonspecific adsorption without compromising the biological integrity of the sample. In this review we highlight why surface attachments are a critical aspect of many single-molecule studies and we discuss current methods for anchoring biomolecules. Finally, we provide a detailed description of a new method developed by our laboratory for anchoring and organizing hundreds of individual DNA molecules on a surface, allowing "high-throughput" studies of protein-DNA interactions at the single-molecule level.

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A single myosin head moves along an actin filament with regular steps of 5.3 nanometres

Cited by 529 publications

References 49 publications

The loose coupling mechanism in molecular machines of living cells

The loose coupling mechanism in molecular machines of living cells

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

The importance of surfaces in single-molecule bioscience

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