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ABSTRACT: F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities.
Nature Communications 08/2012; 3:1022. · 7.40 Impact Factor
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ABSTRACT: Sub-diffraction-limited imaging of fluorescent monomers on sliding microtubules in vitro by nanoscale localization sampling (NLS) is reported. NLS is based on periodic nanohole antenna arrays that create locally amplified electromagnetic hot spots through surface plasmon localization. The localized near-field hot spot temporally samples microtubular movement for enhanced spatial resolution. A fourfold improvement in spatial resolution compared to conventional wide-field microscopy is demonstrated. The resolution enhancement is achieved by imaging rhodamine-labeled microtubules that are sampled by the hot spots to provide sub-diffraction-limited images at 76 nm resolution in the direction of movement and 135 nm orthogonally. The intensity distribution produced by the NLS is measured to be broader than that of conventional imaging, which is consistent with the improvement of imaging resolution. Correlation studies between neighboring nanoantennas are also performed. This confirms the possibility of measuring microtubular transport dynamics. NLS can be useful for moving objects that have a high labeling density or for performing fluctuation spectroscopy in small volumes, and may allow "super-resolution on demand" by customizing nanoantenna structures for specific resolution needs.
Small 12/2011; 8(6):892-900, 786. · 8.35 Impact Factor
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ABSTRACT: F(1)-ATPase is a water-soluble portion of F(o)F(1)-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit γ has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit γ between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a ~4° outward tilting of the γ-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the γ-subunit and provides a new constraint condition of the ATP-waiting conformation of F(1)-ATPase, which has not been determined as an atomic structure from x-ray crystallography.
Biophysical Journal 11/2011; 101(9):2201-6. · 3.65 Impact Factor
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ABSTRACT: F(1)-ATPase is the smallest rotary molecular motor ever found. Unidirectional rotation of the γ-shaft is driven by precisely coordinated sequential ATP hydrolysis reactions in three catalytic sites arranged 120° apart in the cylinder. Single-molecule observation allows us to directly watch the rotation of the shaft using micron-sized plastic beads. Additionally, an advanced version of "total internal reflection fluorescence microscope (TIRFM)" enables us to detect binding and release of energy currency through fluorescently labeled ATP. In this chapter, we describe how to set up the system for simultaneous observation of these two critical events. This specialized optical setup is applicable to a variety of research, not only molecular motors but also other single-molecule topics.
Methods in molecular biology (Clifton, N.J.) 01/2011; 778:259-71.
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ABSTRACT: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.
Biochemical and Biophysical Research Communications 01/2011; 405(4):638-43. · 2.48 Impact Factor
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Takayuki Nishizaka
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ABSTRACT: The final goal of our group is to establish the missing link between chemical reaction and mechanical event in molecular motors. To achieve this, we have developed advanced versions of conventional optical microscopes and applied them into single-molecule techniques. In this chapter we present two studies: one is about the kinesin-microtubule system and the other F(1)-ATPase. These techniques are applicable to other molecular machines, hopefully in more sophisticated ways, and we hope to investigate this in future studies.
Advances in biochemical engineering/biotechnology 03/2010; 119:3-15. · 1.64 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 12/2009; 54(15):1919-27.
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ABSTRACT: Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non-cargo proteins during COPII vesicle formation using single-molecule microscopy combined with an artificial planar lipid bilayer. Single-molecule analysis showed that the Sar1p-Sec23/24p-cargo complex, but not the Sar1p-Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non-cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non-cargo proteins from the COPII vesicles.
The EMBO Journal 09/2009; 28(21):3279-89. · 9.20 Impact Factor
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ABSTRACT: Rotation of the central shaft gamma subunit in a molecular motor F(1)-ATPase is assumed to correlate with and probably be driven by domain motions of the three catalytic beta subunits. Here we observe directly these beta motions through an attached fluorophore, concomitantly with 80 degrees and 40 degrees substep rotations of gamma in the same single molecules. We show the sequence of conformations that each beta subunit undergoes in three-step bending, a approximately 40 degrees counterclockwise turn followed by two approximately 20 degrees clockwise turns, occurring in synchronization with two substep rotations of gamma. The results indicate that most previous crystal structures mimic the conformational set of three beta subunits in the catalytic dwells. Moreover, a previously undescribed set of beta conformations, open, closed and partially closed, is revealed in the ATP-waiting dwells. The present study thus bridges the gap between the chemical and mechanical steps in F(1)-ATPase.
Nature Structural & Molecular Biology 12/2008; 15(12):1326-33. · 12.71 Impact Factor
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ABSTRACT: Mitotic kinesin Eg5 is a homotetrameric molecular motor that cross-links and slides microtubules. The extent to which Eg5 moves processively is not clear. Here we use three-dimensional tracking of a quantum dot attached to the microtubule in a motility assay to directly visualize the corkscrew motion of a sliding microtubule. We show that the rotational pitch of microtubule sliding conveniently reports on the processivity of the driving motors, confirming that two-headed Eg5 is much less processive than two-headed kinesin-1.
Nature Structural & Molecular Biology 10/2008; 15(10):1119-21. · 12.71 Impact Factor
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ABSTRACT: F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.
Cell 08/2007; 130(2):309-21. · 32.40 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 05/2007; 52(4):317-22.
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ABSTRACT: Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P(i) in its individual actions at a single molecule level. The F(1) portion of ATP synthase, also called F(1)-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of DeltaG(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.
Biophysical Journal 04/2007; 92(5):1806-12. · 3.65 Impact Factor
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ABSTRACT: F(o)F(1)-ATP synthase catalyzes the synthesis of ATP using proton-motive force across a membrane. When isolated, the F1 sector, composed of five polypeptide chains with a stoichiometry of alpha(3)beta(3)gammadeltaepsilon, solely hydrolyzes ATP into ADP and phosphate, and is thus called F(1)-ATPase. Rotation of a shaft domain in F(o)F(1)-ATP synthase has been hypothesized by Paul Boyer, and ultimately was confirmed by direct observation as rotation of the gamma-subunit in an isolated alpha(3)beta(3)gamma subcomplex. Unitary turnover of ATP induces 120 degrees steps, consistent with the configuration of three catalytic sites arranged 120 degrees apart around gamma. We have shown the relationships between chemical and mechanical events by imaging individual F(1) molecules under an optical microscope. A new scheme emerges: as soon as a catalytic site binds ATP, the gamma-subunit always turns the same face (interaction surface) to the beta hosting that site; approximately 80 degrees rotation is driven by ATP binding; approximately 40 degrees rotation is induced by completion of hydrolysis [and/or phosphate release] in the site that bound ATP one step earlier.
Methods in molecular biology (Clifton, N.J.) 02/2007; 392:171-81.
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ABSTRACT: F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.
Nature Structural & Molecular Biology 03/2004; 11(2):142-8. · 12.71 Impact Factor
