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 F_oF₁-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.     

Molecular Dynamics Simulations of the Rotary Motor F0 under External Electric Fields across the Membrane

The membrane-bound component F₀, which is a major component of the F₀F₁-ATP synthase, works as a rotary motor and plays a central role in driving the F₁ component to transform chemiosmotic energy into ATP synthesis. We conducted molecular dynamics simulations of b₂-free F₀ in a 1-palmitoyl-2-oleoyl-phosphatidylcholine lipid bilayer for tens of nanoseconds with two different protonation states of the cAsp-61 residue at the interface of the a-c complex in the absence of electric fields and under electric fields of ±0.03 V/nm across the membrane. To our surprise, we observed that the upper half of the N-terminal helix of the c₁ subunit rotated about its axis clockwise by 30°. An energetic analysis revealed that the electrostatic repulsion between this N-terminal helix and subunit c₁₂ was a major contributor to the observed rotation. A correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. The deuterium order parameter (S_CD) profile calculated by averaging all the lipids in the F₀-bound bilayer was not very different from that of the pure bilayer system, in agreement with recent ²H solid-state NMR experiments. However, by delineating the lipid properties according to their vicinity to F₀, we found that the S_CD profiles of different lipid shells were prominently different. Lipids close to F₀ formed a more ordered structure. Similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. The lipids in the proximity of F₀ exhibited very significantly reduced diffusional motion. The numerical value of S_CD was anticorrelated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. Our findings will help elucidate the dynamics of F₀ depending on the protonation state and electric field, and may also shed some light on the interactions between the motor F₀ and its surrounding lipids under physiological conditions, which could help to rationalize its extraordinary energy conversion efficiency.     

Resonance phenomenon of the ATP motor as an ultrasensitive biosensor

We designed a rotary biosensor as a damping effector, with the rotation of the F₀F₁-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection.     

Effect of external torque on the ATP-driven rotation of F1-ATPase

F₁-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F₀ to synthesize ATP in vivo. Therefore elucidation of the behavior of F₁ under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F₁ rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 μM), 120° stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.     

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3

F₁-ATPase is the world’s smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F₁-ATPase (F₁) from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F₁ molecule that consists of one mutant β and two wild-type βs (hybrid F₁). As a result, the new hybrid F₁ showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

Mechanical Modulation of ATP-binding Affinity of V1-ATPase

V₁-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F₁-ATPase (the closest relative of V₁-ATPase evolutionarily), the role of ATP binding for V₁-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V₁-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V₁-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V₁-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k_on) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k_off) was exponentially reduced. The angle dependence of the k_off of V₁-ATPase was significantly smaller than that of F₁-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V₁-ATPase. When V₁-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k_on was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V₁-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.     

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F₁-ATPase (F₁) by attaching probes with different viscous drag coefficients at the rotary shaft of F₁. Individual rotation pauses of F₁ between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F₁ rate enhancement.     

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F₁-ATPase (F₁) by attaching probes with different viscous drag coefficients at the rotary shaft of F₁. Individual rotation pauses of F₁ between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F₁ rate enhancement.     

Robustness of the Rotary Catalysis Mechanism of F1-ATPase

F₁-ATPase (F₁) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F₁ as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F₁ is far more robust than previously thought.     

Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation

F₁-ATPase (F₁) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F₁ produces ATP against the chemical potential of ATP hydrolysis, suggesting that F₁ modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F₁ has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle. Here, we report direct evidence that the ATP-binding rate of F₁ increases upon the forward rotation of the rotor, and its binding affinity to ATP is enhanced by rotation.     

The effects of external electric field: creating non-zero first hyperpolarizability for centrosymmetric benzene and strongly enhancing first hyperpolarizability for non-centrosymmetric edge-modified graphene ribbon H2N-(3,3)ZGNR-NO2

How to generate a non-zero first hyperpolarizability for a centrosymmetric molecule is a challenging question. In this paper, an external (pump) electric field is used to make a centrosymmetric benzene molecule generate a non-zero value of the electric field induced first hyperpolarizability (β ^F ). This comes from the centrosymmetry breaking of electron cloud. Two interesting rules are exhibited. (1) β ^F is anisotropic for different directional fields (F _i, i?=?X, Y, Z). (2) The field dependence of β ^F is a non-monotonic function, and an optimum external electric field causes the maximum value of β ^F . The largest first hyperpolarizability β ^F reaches the considerable level of 3.9?×?10⁵ a.u. under F _Y?=?330?×?10^?4 a.u. for benzene. The external electric field effects on non-centrosymmetric edge-modified graphene ribbon H₂N-(3,3)ZGNR-NO₂ was also studied in this work. The first hyperpolarizability reaches as much as 2.1?×?10⁷ a.u. under F _X?=?600?×?10^?4 a.u. for H₂N-(3,3)ZGNR-NO₂. We show that the external electric field can not only create a non-zero first hyperpolarizability for centrosymmetric molecule, but also remarkably enhance the first hyperpolarizability for a non-centrosymmetric molecule.     

Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+-ATPase by cloned genes from the same and different species

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha-subunit gene of the F₁F₀ type of H⁺-ATPase was deleted. MS117 had low H⁺-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha-subunit gene of E. hirae H⁺-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F₁F₀-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H⁺-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F₁F₀ type of H⁺-ATPase.     

Reversible control of F1-ATPase rotational motion using a photochromic ATP analog at the single molecule level

Motor enzymes such as F₁-ATPase and kinesin utilize energy from ATP for their motion. Molecular motions of these enzymes are critical to their catalytic mechanisms and were analyzed thoroughly using a single molecule observation technique. As a tool to analyze and control the ATP-driven motor enzyme motion, we recently synthesized a photoresponsive ATP analog with a p-tert-butylazobenzene tethered to the 2′ position of the ribose ring. Using cis/trans isomerization of the azobenzene moiety, we achieved a successful reversible photochromic control over a kinesin-microtubule system in an in vitro motility assay. Here we succeeded to control the hydrolytic activity and rotation of the rotary motor enzyme, F₁-ATPase, using this photosensitive ATP analog. Subsequent single molecule observations indicated a unique pause occurring at the ATP binding angle position in the presence of cis form of the analog.     

The nonlinear chemo-mechanic coupled dynamics of the F 1 -ATPase molecular motor

The ATP synthase consists of two opposing rotary motors, F₀ and F₁, coupled to each other. When the F₁ motor is not coupled to the F₀ motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F₁-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F₁-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F₁-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F₁ motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F₁ motor, which increases the motor’s rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F₁ motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F₁ motor and provide insight about the stochastic wave mechanics of F₁-ATPase.     

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD⁺+NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD⁺+NADH both were added. The change was lacking in the presence of the F₀F₁-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F₀F₁-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K⁺-dependent F₀F₁-ATPase activity, suggesting a relationship between the F₀F₁-ATPase and K⁺ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

The proton-driven ATP synthase (F_OF₁) is comprised of two rotary, stepping motors (F_O and F₁) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded F_O c-ring. Proton transport by F_O is firmly coupled to the rotation of the c-ring relative to other F_O subunits (ab₂). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.     

Net ATP synthesis in H+ -atpase macroliposomes by an external electric field.

Application of electric pulses (1000 V/cm, 20 m sec duration) to macroliposomes containing pure stable H⁺-ATPase (F₀·F₁) resulted in synthesis of ATP. Microliposomes containing F₀·F₁ showed very little ATP synthesis under the same conditions. The amount of ATP synthesized was increased by increasing the number of electric pulses applied and decreased by addition of either an uncoupler or an energy transfer inhibitor.     

A rapidly new-typed detection of norovirus based on F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATPase molecular motor biosensor

In order to adapt port rapid detection of food borne norovirus, presently we developed a new typed detection method based on F₀F₁-ATPase molecular motor biosensor. A specific probe was encompassed the conservative region of norovirus and F₀F₁-ATPase within chromatophore was constructed as a molecular motor biosensor through the “ε-subunit antibody-streptomycin-biotin-probe” system. Norovirus was captured based on probe-RNA specific binding. Our results demonstrated that the Limit of Quantification (LOQ) is 0.005 ng/mL for NV RNA and also demonstrated that this method possesses specificity and none cross-reaction for food borne virus. What’s more, the experiment used this method could be accomplished in 1 h. We detected 10 samples by using this method and the results were consistent with RT-PCR results. Overall, based on F₀F₁-ATPase molecular motors biosensor system we firstly established a new typed detection method for norovirus detection and demonstrated that this method is sensitive and specific and can be used in the rapid detection for food borne virus.