Rotation Of F1-ATPase Research Articles

A Change in the Radius of Rotation of F1-ATPase Indicates a Tilting Motion of the Central Shaft

F1-ATPase is a water-soluble portion of FoF1-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 F1-ATPase, which has not been determined as an atomic structure from x-ray crystallography.

1L1348 表面プラズモン共鳴を引き起こす金微粒子を用いた蛍光ヌクレオチドとF1-ATPaseの回転の同時観察

1L1348 表面プラズモン共鳴を引き起こす金微粒子を用いた蛍光ヌクレオチドとF1-ATPaseの回転の同時観察

Fluctuation Theorem Applied to F1-ATPase

The fluctuation theorem (FT), which is one of fluctuation theories based on non-equilibrium statistical mechanics and represents the property of an entropy production in a small system, was experimentally verified in a motor protein F1-ATPase (F1). The theorem has been applied to several experimental systems such as colloidal particle systems and RNA hairpins. Those systems were in non-equilibrium when the operations were added to the systems and the entropy productions were measured for those non-equilibrium processes. Unlike those systems, F1 is an autonomously non-equilibrium system in which the rotor γ subunit rotates in the stator α3β3 ring upon ATP hydrolysis. Can FT be applied to such an autonomous system? Noting that the entropy production of the probe visualizing the rotation of F1 is a product of the rotary torque and the angular velocity, we introduced the representation of FT appropriate for the torque measurement. The torque measured through our method was compared with that measured conventionally. In addition to the verification, we applied the theorem to a mutant F1 and another motor protein V1. The applicability of FT should be expanded to the wide range of biological systems in vitro and in vivo.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Biomolecular Nano-Flow-Sensor to Measure Near-Surface Flow

We have proposed and experimentally demonstrated that the measurement of the near-surface flow at the interface between a liquid and solid using a 10 nm-sized biomolecular motor of F1-ATPase as a nano-flow-sensor. For this purpose, we developed a microfluidic test-bed chip to precisely control the liquid flow acting on the F1-ATPase. In order to visualize the rotation of F1-ATPase, several hundreds nanometer-sized particle was immobilized at the rotational axis of F1-ATPase to enhance the rotation to be detected by optical microscopy. The rotational motion of F1-ATPase, which was immobilized on an inner surface of the test-bed chip, was measured to obtain the correlation between the near-surface flow and the rotation speed of F1-ATPase. As a result, we obtained the relationship that the rotation speed of F1-ATPase was linearly decelerated with increasing flow velocity. The mechanism of the correlation between the rotation speed and the near-surface flow remains unclear, however the concept to use biomolecule as a nano-flow-sensor was proofed successfully.(See supplementary material 1)

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell

The temperature-dependent rotation of F1-ATPase gamma subunit was observed in V(max) conditions at low viscous drag using a 60-nm gold bead (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). The Arrhenius slopes of the speed of the individual 120 degrees steps and reciprocal of the pause length between rotation steps were very similar, indicating a flat energy pathway followed by the rotationally coupled catalytic cycle. In contrast, the Arrhenius slope of the reciprocal pause length of the gammaM23K mutant F1 was significantly increased, whereas that of the rotation rate was similar to wild type. The effects of the rotor gammaM23K substitution and the counteracting effects of betaE381D mutation in the interacting stator subunits demonstrate that the rotor-stator interactions play critical roles in the utilization of stored elastic energy. The gammaM23K enzyme must overcome an abrupt activation energy barrier, forcing it onto a less favored pathway that results in uncoupling catalysis from rotation.

How is the Temperature Sensitive (TS) Reaction of F1-ATPase Coupled with its Rotation?

F1-ATPase (F1) is a rotary molecular motor in which γ subunit rotates against α3β3 cylinder. So far, 80° and 40° substeps in rotation of F1 have been observed, and kinetic analysis has shown that the 80° substep is initiated by concomitant ATP binding and ADP release, and the 40° substep occurs after ATP hydrolysis and inorganic phosphate release. Temperature sensitive (TS) reaction of F1 has found recently at the ATP binding angle at the temperature below 10 °C, and it has been suggested to correspond to ADP release (Watanabe et al., EMBO report 2008). In this study, TS reaction of βE190D mutant, that has much slower ATP hydrolysis rate than wild type, was found below 20 °C. Q10 factor of TS reaction of βE190D mutant was 16, that is comparable to that of wild type. Previous single-molecule rotation observations of the hybrid F1 that has only one mutant β subunit, α3β(WT)2β(E190D)γ have shown that the β subunit binds ATP at 0°, cleaves ATP at 200°, and release ADP and inorganic phosphate after cleavage of ATP (Ariga et al., NSMB 2007). In contrast to our expectation, observation of hybrid F1 α3β(WT)2β(E190D)γ rotation at 18 °C has revealed that TS reaction occurs at ATP binding angle(0°) to mutated β subunit. Furthermore, simultaneous observation at the video rate (30 frames per seconds) showed that Cy3-ATP bindings and steps in rotation occur concomitantly. We will discuss about the reaction schemes that can explain these results.

Stepwise Propagation of the ATP-induced Conformational Change of the F1-ATPase β Subunit Revealed by NMR

The rotation of F1-ATPase (F1) is driven by the open/close bending motion of the beta subunit. The mechanism underlying the bending motion was investigated for the F1beta monomer from thermophilic Bacillus PS3 (TF1beta) in solution, using mutagenesis and NMR. The hydrogen bond networks involving the side chains of Lys-164 (numbering for TF1beta; 162 for mitochondrial F1beta in parentheses), Thr-165(163), Arg-191(189), Asp-252(256), Asp-311(315), and Arg-333(337) in the catalytic region are significantly different for the ligand-bound and freebeta subunits in the crystal structures of mitochondrial F1. The role of each amino acid residue was examined by Ala substitution. beta(K164A) reduced the affinity constant for 5'-adenyl-beta,gamma-imidodiphosphate by 20-fold and abolished the conformational change associated with nucleotide binding and the ATPase activity of alpha3beta(K164A)3gamma.beta(T165A) and beta(D252A) exhibited no effect on the binding affinity but abolished the conformational change and the ATPase activity. The chemical shift perturbation of backbone amide signals of the segmentally labeled beta(mutant)s indicated stepwise propagation of the open/close conversion on ligand binding. The key action in the conversion is the switching of the hydrogen-bonding partner of Asp-252 from Lys-164 to Thr-165. Residual dipolar coupling analysis revealed that the closed conformation of the beta monomer was more closed than that in the crystal structure and was different for MgATP- and MgADP-bound beta subunits. Actually, MgATP induced a conformational change around Tyr-307 (311 for MF1beta), whereas MgADP did not. The significance of these findings is discussed in connection with the catalytic rotation of F1-ATPase.

S1/2 Mechanistic insights of F1-ATPase rotation from single-molecule measurements of the power stroke

S1/2 Mechanistic insights of F1-ATPase rotation from single-molecule measurements of the power stroke

Axle-Less F 1 -ATPase Rotates in the Correct Direction

F1-adenosine triphosphatase (ATPase) is an ATP-driven rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of three alpha and three beta subunits alternately arranged. The rotor shaft, an antiparallel alpha-helical coiled coil of the amino and carboxyl termini of the gamma subunit, deeply penetrates the central cavity of the stator cylinder. We truncated the shaft step by step until the remaining rotor head would be outside the cavity and simply sat on the concave entrance of the stator orifice. All truncation mutants rotated in the correct direction, implying torque generation, although the average rotary speeds were low and short mutants exhibited moments of irregular motion. Neither a fixed pivot nor a rigid axle was needed for rotation of F1-ATPase.

Chemical delivery microsystem for single-molecule analysis using multilaminar continuous flow

The fluidic operations, such as chemical delivery, hydrodynamic force control, solutions exchange, etc., to a desired location in a microfluidic channel are one of the most important technical aspects to progress single-molecule studies in microfluidic devices. This paper experimentally demonstrates multilaminar flow-based fluidic operations to supply and remove chemicals to an immobilized single-molecule on an inner surface of a microchannel to develop a future platform microfluidic device for single-molecule analysis. The microfluidic device, having multiple inlets and single outlet embedded with a micropump for each inlet, can generate continuous multiple laminar flow inside of the microchannel. The multiple laminar flow control capabilities were experimentally evaluated to deliver any single laminar flow to a desired location and to control flow speed to minimize hydrodynamic force to the target single-molecule. As a demonstration of single-molecule analysis using this format, ATP dependent rotation of F 1-ATPase was investigated. As a result, the rotation of F1-ATPase was successfully investigated. This format showed many advantages and flexibility to realize the future platform of single-molecule studies.

Gold Functionalized Nano-Needles for Angular Protein Movement Visualization

We have investigated and compared various methods of fabricating silicon nano-needles of 100–200 nm in diameter and 1–2 µm in length for visualization of motor protein movement. Owing to their thin and long geometry, the needles are ideal to amplify and visualize angular movement. To enable highly localized protein attachment, a well-defined attachment point at one end of the needles was prepared. Fabrication by electron-beam lithography as well as by a highly parallel non-lithographic process were implemented and compared. Sensitive angular motion amplification was demonstrated by attachment of needles to F1 ATPase rotation motor proteins. In this report we characterize the fabrication processes, discuss the differences, and present the results of motor protein motion visualization.

Conformational Change of H+-ATPase β Monomer Revealed on Segmental Isotope Labeling NMR Spectroscopy

F1-ATPase has been shown to be a stepwise molecular motor. Its rotation mechanism has been explained by the interaction of the gamma axis with the open and closed forms of the beta subunit. Although NMR should be a powerful method for elucidating its mechanism, its molecular size (473 amino acid residues, 52 kDa) is a major obstacle. We have applied segmental labeling based on intein ligation to the beta subunit, and succeeded in assigning 89% of the NH (402/451), 89% of the Calpha (417/473), 83% of the Cbeta (357/431), and 90% of the CO (425/473) signals of the beta subunit monomer. The secondary structures predicted from the chemical shifts of the main chain atoms and the relative orientations determined from residual dipolar couplings indicated that the subunit beta monomer takes on the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding show that conformational change from the open to the closed form takes place on nucleotide binding. The intrinsic conformational change of the beta subunit monomer induced by nucleotide binding must be one of the essential driving forces for the rotation of F1-ATPase.

Inverse Regulation of Rotation of F1-ATPase by the Mutation at the Regulatory Region on the γ Subunit of Chloroplast ATP Synthase

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Stepping Rotation of F1-ATPase with One, Two, or Three Altered Catalytic Sites That Bind ATP Only Slowly

F(1)-ATPase is an ATP hydrolysis-driven motor in which the gamma subunit rotates in the stator cylinder alpha(3)beta(3). To know the coordination of three catalytic beta subunits during catalysis, hybrid F(1)-ATPases, each containing one, two, or three "slow" mutant beta subunits that bind ATP very slowly, were prepared, and the rotations were observed with a single molecule level. Each hybrid made one, two, or three steps per 360 degrees revolution, respectively, at 5 microm ATP where the wild-type enzyme rotated continuously without step under the same observing conditions. The observed dwell times of the steps are explained by the slow binding rate of ATP. Except for the steps, properties of rotation, such as the torque forces exerted during rotary movement, were not significantly changed from those of the wild-type enzyme. Thus, it appears that the presence of the slow beta subunit(s) does not seriously affect other normal beta subunit(s) in the same F(1)-ATPase molecule and that the order of sequential catalytic events is faithfully maintained even when ATP binding to one or two of the catalytic sites is retarded.

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.

Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.

Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging.

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F(1)-ATPase revealed that the subunit rotates in the molecule in discrete 120 degrees steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120 degrees stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

Direct observation of the rotation of F1-ATPase.

Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells. It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies. We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly. In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary torque produced reached more than 40 pN nm(-1) under high load.