Simultaneous Observation of Individual ATPase and Mechanical Events by a Single Myosin Molecule during Interaction with Actin

Ishijima, Akihiko; Kojima, Hiroaki; Funatsu, Takashi; Tokunaga, Makio; Higuchi, Hideo; Tanaka, Hiroto; Yanagida, Toshio

doi:10.1016/s0092-8674(00)80911-3

Cited by 465 publications

(351 citation statements)

References 55 publications

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“…M5SHs were copolymerized into long (6.6 m on average) filaments with rabbit skeletal muscle myosin rods without heads. The total concentration of the proteins was set to be 0.15 M, and the molar ratio of the M5SH to myosin rod in the mixture was adjusted to be 1:2,000 so that only a small number of M5SHs were incorporated into a cofilament (9,16).…”

Section: Recombinant M5sh and Two-headed Myosin-v (M5dh)mentioning

confidence: 99%

“…To attach beads to the two ends of an actin filament, the surface of polystyrene latex beads (0.945 m in diameter) was coated with ␣-actinin by means of (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; see Supporting Text, which is published as supporting information on the PNAS web site). Cofilaments applied to a flow chamber with pedestals on a glass slide surface were adsorbed onto the pedestal surface (9,16). The pedestal surface was coated with casein to prevent ␣-actinin-coated beads from nonspecifically binding to the glass surface.…”

Section: Recombinant M5sh and Two-headed Myosin-v (M5dh)mentioning

confidence: 99%

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A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

Watanabe

Tanaka

Iwane

et al. 2004

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

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Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large (Ϸ36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP-and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (Ϸ32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.C lass V myosin (myosin-V) is a linear motor protein that moves unidirectionally along an actin filament with successive large (Ϸ36-nm) steps (1-3). Myosin-V has two heads, each of which consists of a motor domain and a long neck domain. Based on this structural feature, a ''hand-over-hand'' mechanism has been proposed to explain a unidirectional and successive large stepping of myosin-V (4). The proposed mechanism is that the trailing head detaches upon ATP binding, and rapidly moves forward by Ϸ72 nm (5). Upon reattachment with actin, it becomes the new leading head. The partner and former leading head remains attached with the same actin monomer. Based on a ''lever-arm tilting model'' (6), it has been proposed that the tilting of the long neck domain of the attached leading head biases Brownian motion of the trailing head forward (7). Recently, it was demonstrated by using a single-molecule f luorescence polarization technique that the orientation of f luorophores on calmodulin light chain attached to the neck domain of myosin-V indeed alters before and after mechanical steps (8). This finding is consistent with the lever-arm tilting model. However, it is still unclear whether the observed tilting of the neck domain may not be the cause of steps but rather the effect of steps generated by other mechanisms. This uncertainty is because the time resolution of the f luorescence polarization was not sufficient to detect the change directly coupled to these steps.On the other hand, we have recently reported that a short (Ϸ4-nm) neck mutant of myosin-V can also generate successive large (Ϸ35-nm) steps (9). That observation poses a challenge to the lever-arm tilting model because the neck domain is too short to lead the trailing head to the next target site Ϸ72 nm distant from the original site, especially at high loads. In support of this finding, class-VI myosin, which has a naturally very short (Ϸ4-nm) neck domain, has been found to move processively with large steps as well as myosin-V (10, 11).A central issue for the multiple successive large steps according to the hand-over-hand mechanism based on the lever-arm tilting model is that the two-headed structure is indispensable. P...

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Section: Recombinant M5sh and Two-headed Myosin-v (M5dh)mentioning

confidence: 99%

Section: Recombinant M5sh and Two-headed Myosin-v (M5dh)mentioning

confidence: 99%

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

Watanabe

Tanaka

Iwane

et al. 2004

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

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“…The development reported here expands on the current arsenal of hybrid single molecule techniques combining force and other observables (8,(25)(26)(27). Unlike DNA or RNA hairpins, where forces on the order of 15 pN are necessary to induce mechanical unzipping (10,11), the conformations of HJs could be biased at 0.5 pN or lower.…”

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confidence: 99%

Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

Hohng

Zhou

Nahas

et al. 2007

Science

267

299

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Despite the recent advances in single molecule manipulation techniques, purely mechanical approaches can not detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence which allowed us to examine the effect of sub-pN forces on the nanometer scale motion of the Holliday junction (HJ) at 100 Hz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.Many biological processes are dependent on tension. In recent years, single molecule force measurements have shown directly that biochemical reactions can be influenced by applied force (1). Yet, purely mechanical tools can not detect small scale conformational changes unless strong enough and persistent force is applied. At weak forces, the flexible tether connecting the mechanical probe to the biological molecule is not stretched enough to transmit small movements. This is unfortunate because weak and transient forces are likely more prevalent in vivo but the experimental limitations confine single molecule mechanical studies to examining the effect of relatively large forces. We aimed to study the effect of weak external forces on the biomolecular conformational dynamics by combining single molecule fluorescence resonance energy transfer (smFRET) (2-4) with manipulation using optical trap (5). smFRET has high spatial resolution (≤ 5 Å) (6, 7)) and can be measured at arbitrarily low forces. Previous attempts to combine FRET and optical trap using the DNA hairpin as a model system (8, 9) did not reveal new information because the hairpin unzips at high forces (~ 15 pico Newton (pN)), a regime that had been extensively investigated using force-based techniques (10, 11). Here, we report an approach to detect nanometer-* To whom correspondence should be addressed. (tjha@uiuc.edu). † Present Address: Department of Physics and Astronomy, Seoul National University, Seoul 151-742, Korea. scale motion at sub-pN forces. We used the approach to gain insight into the reaction landscape of the Holliday junction (HJ) by gently stretching it along different directions. NIH Public AccessThe HJ is a four-stranded DNA structure that forms as an intermediate during recombination (12). To understand the mechanisms of cellular enzymes that function with the HJ, a detailed description of the static and dynamic structural properties of the HJ itself is needed. In the absence of added ions the HJ adopts an open structure, where the four helical arms point toward the corners of a square (13, 14) (Fig. 1A). In the presence ...

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“…Myosins motor domains are ideal model systems for developing and advancing protein engineering and design because they produce mechanical changes that can be directly followed by a large variety of techniques, in addition to spectroscopic signals and chemical changes during their catalytic cycle. This facilitates the testing and direct confirmation of the correct stereospecific attachment of engineered modules using in vitro motility assays and single molecule approaches (Kron & Spudich 1986;Finer et al 1994;Molloy et al 1995a;Ishijima et al 1998).…”

Section: Resultsmentioning

confidence: 99%

Molecular engineering of myosin

Manstein

2004

Phil. Trans. R. Soc. Lond. B

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Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin-and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins.

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Simultaneous Observation of Individual ATPase and Mechanical Events by a Single Myosin Molecule during Interaction with Actin

Cited by 465 publications

References 55 publications

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

Molecular engineering of myosin

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