None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

F₁-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α₃β₃γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α₃β₃ subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F₁, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F₁ is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.     

Torque Generation Mechanism of F1-ATPase upon NTP Binding

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F₁-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10³ and 10⁶ times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F₁ to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F₁ exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F₁ with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.     

Theoretical Analysis of the F1-ATPase Experimental Data

F₁-ATPase is a rotatory molecular motor fueled by ATP nucleotides. Different loads can be attached to the motor axis to show that it rotates in main discrete steps of 120° with substeps of ∼80° and 40°. Experimental data show the dependence on the mean rotational velocity ω with respect to the external control parameters: the nucleotide concentration [ATP] and the friction of the load γ_L. In this work we present a theoretical analysis of the experimental data whose main results are: 1), A derivation of a simple analytical formula for ω([ATP], γ_L) that compares favorably with experiments; 2), The introduction of a two-state flashing ratchet model that exhibits experimental phenomenology of a greater specificity than has been, to our knowledge, previously available; 3), The derivation of an argument to obtain the values of the substep sizes; 4), An analysis of the energy constraints of the model; and 5), The theoretical analysis of the coupling ratio between the ATP consumed and the success of a forward step. We also discuss the compatibility of our approach with recent experimental observations.     

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.     

Origin of apparent negative cooperativity of F1-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F₁-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F₁-ATPase, α_(W463F)3β_(Y341W)3γ and α_{(K175A/T176A/W463F)3}β_(Y341W)3γ. For α_(W463F)3β_(Y341W)3γ, apparent K_m's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent K_m's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent K_m of ATPase activity (10 μM) roughly agreed with the highest K_m of fluorescence measurements (27 μM). The results indicate that in case of α_(W463F)3β_(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K_m of ATPase activity does not represent the ATP binding to a catalytic site. In case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, the K_m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α_{(K175A/T176A/W463F)3}β_(Y341W)3γ was rather linear compared with that of α_(W463F)3β_(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F₁-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K_m's of 100-300 μM and 1-30 μM range for wild-type F₁-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

The coupled chemomechanics of the F1-ATPase molecular motor

The enzyme F₁-ATPase is a rotary nanomotor in which the central γ subunit rotates inside the cavity made of α₃β₃ subunits. The experiments showed that the rotation proceeds in steps of 120° and each 120° step consists of 80° and 40° substeps. Here the Author proposes a stochastic wave mechanics of the F₁-ATPase motor and combines it with the structure-based kinetics of the F₁-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F₁ motor. Using the model, several rate-limited situations about γ subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.     

On chemomechanical coupling of the F1-ATPase molecular motor

F₁-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F₁-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F₁-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well.     

Stimulation of F1-ATPase activity by sodium dodecyl sulfate

F₁-ATPase is a rotary molecular motor in which the γ subunit rotates inside the cylinder made of α₃β₃ subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F₁-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2 mM ATP. Maximal stimulation was obtained at 0.003% (w/v) SDS, the initial (least inhibited) activity being about 1.4 times and the steady-state activity 3-4 times the values in the absence of SDS. Rotation rates observed with a 40-nm gold bead or a 0.29-μm bead duplex as well as the torque were unaffected by the presence of 0.003% SDS. The fraction of beads that rotated, in contrast, tended to increase in the presence of SDS. SDS seems to bring inactive F₁ molecules into an active form but it does not alter or enhance the function of already active F₁ molecules significantly.     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F₁-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF₁-ATPase. The molecular weights of pea mitochondrial F₁-ATPase and pea chloroplast CF₁-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F₁-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF₁-ATPase. The purified mitochondrial F₁-ATPase exhibited coupling factor activity. In spite of the observed differences between CF₁ and F₁, the mitochondrial enzyme stimulated ATP formation in CF₁-depleted pea chloroplast membranes. Thus, the mitochondrial F₁ was able to substitute functionally for the chloroplast CF₁ in reconstituting photophosphorylation.     

Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles

Bovine heart submitochondrial particles depleted of F₁ by treatment with urea (‘F₁-depleted particles’) were incubated with soluble F₁-ATPase. The binding of F₁ to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH⁺₄, K⁺, Rb⁺, Cs⁺, Na⁺ and Li⁺ promoted reconstitution maximally at 40–74 mM, guanidinium⁺ and Tris⁺ at 20–30 mM, and Ca²⁺ and Mg²⁺ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F₁ binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F₁-depleted thylakoid membranes and with submitochondrial particles depleted of both F₁ and the coupling proteins F₆ and oligomycin sensitivity-conferring protein.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F₁ extracted from these particles. All of these agents cause inhibition of ATPase in F₁ as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F₁. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F₁ complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

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.     

Purification and properties of the latent F1-ATPase of Micrococcus lysodeikticus

The latent coupling factor (F₁)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F₁ from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits α, β, γ, δ, and ? and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with ¹⁴C-labelled, F₁-ATPase prepared from cells grown on medium containing [U-¹⁴C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3.     

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.     

Classification of nucleotide binding sites on mitochondrial F1-ATPase from yeast

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F₁ from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.     

高活性F1-ATP酶单分子旋转初步观察

从基因突变的F₁-ATP酶（基因突变质粒,α-C193S, γ-S107C,β亚基带有10个组氨酸标记（His-Tag）,转入到菌株大肠杆菌JM103）的菌株中筛选出一高表达菌株.该菌株表达的F₁-ATP酶经纯化后其水解活性明显高于文献值. 从单分子水平上进行观察,发现在水解ATP过程中,γ亚基上连接的荧光标记蛋白微丝,其旋转速度要比文献中同样条件下快约一倍.