Nonequilibrium dissipation-free transport in F?-ATPase and the thermodynamic role of asymmetric allosterism.
ABSTRACT: F1-ATPase (or F1), the highly efficient and reversible biochemical engine, has motivated physicists as well as biologists to imagine the design principles governing machines in the fluctuating world. Recent experiments have clarified yet another interesting property of F1; the dissipative heat inside the motor is very small, irrespective of the velocity of rotation and energy transport. Conceptual interest is devoted to the fact that the amount of internal dissipation is not simply determined by the sequence of equilibrium pictures, but also relies on the rotational-angular dependence of nucleotide affinity, which is a truly nonequilibrium aspect. We propose that the totally asymmetric allosteric model (TASAM), where adenosine triphosphate (ATP) binding to F1 is assumed to have low dependence on the angle of the rotating shaft, produces results that are most consistent with the experiments. Theoretical analysis proves the crucial role of two time scales in the model, which explains the universal mechanism to produce the internal dissipation-free feature. The model reproduces the characteristic torque dependence of the rotational velocity of F1 and predicts that the internal dissipation upon the ATP synthesis direction rotation becomes large at the low nucleotide condition.
Project description:We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of FoF1-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F1-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼ 1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than k(B)T per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼ 1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in Fo and F1, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.
Project description:F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).
Project description: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).
Project description: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 ?-phosphate of ATP (general base); and an arginine directly contacting the ?-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.
Project description:F1-ATPase (F1) is a rotary motor protein that couples ATP hydrolysis to mechanical rotation with high efficiency. In our recent study, we observed a highly temperature-sensitive (TS) step in the reaction catalyzed by a thermophilic F1 that was characterized by a rate constant remarkably sensitive to temperature and had a Q10 factor of 6-19. Since reactions with high Q10 values are considered to involve large conformational changes, we speculated that the TS reaction plays a key role in the rotation of F1. To clarify the role of the TS reaction, in this study, we conducted a stall and release experiment using magnetic tweezers, and assessed the torque generated during the TS reaction. The results indicate that the TS reaction generates the same amount of rotational torque as does ATP binding, but more than that generated during ATP hydrolysis. Thus, we confirmed that the TS reaction contributes significantly to the rotation of F1.
Project description:FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.
Project description: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.
Project description:Inspired by the dynamics of the dissipative self-assembly of microtubules, chemically fueled synthetic systems with transient lifetimes are emerging for nonequilibrium materials design. However, realizing programmable or even adaptive structural dynamics has proven challenging because it requires synchronization of energy uptake and dissipation events within true steady states, which remains difficult to orthogonally control in supramolecular systems. Here, we demonstrate full synchronization of both events by ATP-fueled activation and dynamization of covalent DNA bonds via an enzymatic reaction network of concurrent ligation and cleavage. Critically, the average bond ratio and the frequency of bond exchange are imprinted into the energy dissipation kinetics of the network and tunable through its constituents. We introduce temporally and structurally programmable dynamics by polymerization of transient, dynamic covalent DNA polymers with adaptive steady-state properties in dependence of ATP fuel and enzyme concentrations. This approach enables generic access to nonequilibrium soft matter systems with adaptive and programmable dynamics.
Project description:Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ? subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058-42067). In this study, we constructed a series of ? subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ? subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ?Ser(108) in loop 2 and ?Tyr(114) in helix 2, which possibly interact with the ? and ? subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ? subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits.
Project description:F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1-3.4?Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of Fo associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F1 and Fo motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the Fo motor.