Design of artificial molecular motor inheriting directionality and scalability.

Realizing artificial molecular motors with autonomous functionality and high performance is a major challenge in biophysics. Such motors not only provide new perspectives in biotechnology but also offer a novel approach for the bottom-up elucidation of biological molecular motors. Directionality and scalability are critical factors for practical applications. However, the simultaneous realization of both remains challenging. In this study, we propose a novel design for a rotary motor that can be fabricated using a currently available technology, DNA origami, and validate its functionality through simulations with practical parameters. We demonstrate that the motor rotates unidirectionally and processively in the direction defined by structural asymmetry, which induces kinetic asymmetry in motor motion. The motor also exhibits scalability, such that increasing the number of connections between the motor and stator allows for a larger speed, run length, and stall force.

Error-suppression mechanism of PCR by blocker strands.

The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent applications, including virus tests, DNA sequencing, genotyping, and genome cloning. These applications rely on the specificity of the primer hybridization and therefore require effective suppression of hybridization errors. A simple and effective method to achieve that is to add blocker strands, also called clamps, to the PCR mixture. These strands bind to the unwanted target sequence, thereby blocking the primer mishybridization. Because of its simplicity, this method is applicable to a broad nucleic-acid-based biotechnology. However, the precise mechanism by which blocker strands suppress PCR errors remains to be understood, limiting the applicability of this technique. Here, we combine experiments and theoretical modeling to reveal this mechanism. We find that the blocker strands both energetically destabilize the mishybridized complex and sculpt a kinetic barrier to suppress mishybridization. This combination of energetic and kinetic biasing extends the viable range of annealing temperatures, which reduces design constraint of the primer sequence and extends the applicability of PCR.

Optimal Control of the F1-ATPase Molecular Motor.

F1-ATPase is a rotary molecular motor that in vivo is subject to strong nonequilibrium driving forces. There is great interest in understanding the operational principles governing its high efficiency of free-energy transduction. Here we use a near-equilibrium framework to design a nontrivial control protocol to minimize dissipation in rotating F1 to synthesize adenosine triphosphate. We find that the designed protocol requires much less work than a naive (constant-velocity) protocol across a wide range of protocol durations. Our analysis points to a possible mechanism for energetically efficient driving of F1 in vivo and provides insight into free-energy transduction for a broader class of biomolecular and synthetic machines.

Optimal Rectification without Forward-Current Suppression by Biological Molecular Motor.

We experimentally show that biological molecular motor F_{1}-ATPase (F_{1}) implements an optimal rectification mechanism. The rectification mechanism hardly suppresses the synthesis of adenosine triphosphate by F_{1}, which is F_{1}'s physiological role, while inhibiting the unfavorable hydrolysis of adenosine triphosphate. This optimal rectification contrasts highly with that of a simple ratchet model, where the inhibition of the backward current is inevitably accompanied by the suppression of the forward current. Our detailed analysis of single-molecule trajectories demonstrates a novel but simple rectification mechanism of F_{1} with parallel landscapes and asymmetric transition rates.

Cooperative stator assembly of bacterial flagellar motor mediated by rotation.

Cooperativity has a central place in biological regulation, providing robust and highly-sensitive regulation. The bacterial flagellar motor implements autonomous torque regulation based on the stator's dynamic structure; the stator units bind to and dissociate from the motor dynamically in response to environmental changes. However, the mechanism of this dynamic assembly is not fully understood. Here, we demonstrate the cooperativity in the stator assembly dynamics. The binding is slow at the stalled state, but externally forced rotation as well as driving by motor torque in either direction boosts the stator binding. Hence, once a stator unit binds, it drives the rotor and triggers the avalanche of succeeding bindings. This cooperative mechanism based on nonequilibrium allostery accords with the recently-proposed gear-type coupling between the rotor and stator.

Harnessing random low Reynolds number flow for net migration.

Random noise in low Reynolds number flow has rarely been used to obtain net migration of microscale objects. In this study, we numerically show that net migration of a microscale object can be extracted from random directional fluid forces in Stokes flow, by introducing deformability and inhomogeneous density into the object. We also developed a mathematical framework to describe the deformation-induced migration caused by noise. These results provide a basis for understanding the noise-induced migration of a microswimmer and are useful for harnessing energy from low Reynolds number flow.

Tight Chemomechanical Coupling of the F1 Motor Relies on Structural Stability.

The F1 motor is a rotating molecular motor that ensures a tight chemomechanical coupling between ATP hydrolysis/synthesis reactions and rotation steps. However, the mechanism underlying this tight coupling remains to be elucidated. In this study, we used electrorotation in single-molecule experiments using an F1ßE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F1, indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type. Experiments on the ATPase activity after heat treatment and gel filtration of the α3ß3-subcomplex revealed the unstable structure of the ßE190D mutant. Our results suggest that the tight chemomechanical coupling of the F1 motor relies on the structural stability of F1. We also discuss the difference between the stepping torque and the stall torque.

Efficiencies of molecular motors: a comprehensible overview.

Many biological molecular motors can operate specifically and robustly at the highly fluctuating nano-scale. How these molecules achieve such remarkable functions is an intriguing question that requires various notions and quantifications of efficiency associated with the operations and energy transduction of these nano-machines. Here we give a short review of some important concepts of motor efficiencies, including the thermodynamic, Stokes, and generalized and transport efficiencies, as well as some implications provided by the thermodynamic uncertainty relations recently developed in nonequilibrium physics.

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control.

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.

Assembly of a functional and responsive microstructure by heat bonding of DNA-grafted colloidal brick.

A micromachine constructed to possess various chemical and mechanical functions is one of the ultimate targets of technology. Conventional lithographic processes can be used to form complicated structures. However, they are basically limited to rigid and static structures with poor surface properties. Here, we demonstrate a novel method for assembling responsive and functional microstructures from diverse particles modified with DNA strands. The DNA strands are designed to form hairpins at room temperature and denature when heated. Structures are assembled through the simultaneous manipulation and heating of particles with "hot" optical tweezers, which incorporates the particles one by one. The flexible connection formed by DNA strands allows the responsive deformation of the structures with local controllability of the structural flexibility. We assembled a microscopic robot arm actuated by an external magnet, a hinge structure with a locally controlled connection flexibility and a three-dimensional double helix structure. The method is simple and can also be applied to build complex biological tissues from cells.

Thermodynamic analyses of nucleotide binding to an isolated monomeric ß subunit and the α3ß3γ subcomplex of F1-ATPase.

Rotation of the γ subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a ßY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a ßE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric ßY341W subunit and a mutant α3ß3γ subcomplex containing the ßY341W mutation with or without an additional ßE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated ß subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van't Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated ß subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH(0) and ΔG(0) values at 25°C. The binding of nucleotides with different bases to the isolated ß subunit resulted in different entropy changes. Interestingly, NDP binding to the α3ß(Y341W)3γ subcomplex had similar Kd and ΔG(0) values as binding to the isolated ß(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.

Experimental thermodynamics of single molecular motor.

Molecular motor is a nano-sized chemical engine that converts chemical free energy to mechanical motions. Hence, the energetics is as important as kinetics in order to understand its operation principle. We review experiments to evaluate the thermodynamic properties of a rotational F1-ATPase motor (F1-motor) at a single-molecule level. We show that the F1-motor achieves 100% thermo dynamic efficiency at the stalled state. Furthermore, the motor reduces the internal irreversible heat inside the motor to almost zero and achieves a highly-efficient free energy transduction close to 100% during rotations far from quasistatic process. We discuss the mechanism of how the F1-motor achieves such a high efficiency, which highlights the remarkable property of the nano-sized engine F1-motor.

Information-to-free-energy conversion: Utilizing thermal fluctuations.

Maxwell's demon is a hypothetical creature that can convert information to free energy. A debate that has lasted for more than 100 years has revealed that the demon's operation does not contradict the laws of thermodynamics; hence, the demon can be realized physically. We briefly review the first experimental demonstration of Maxwell's demon of Szilard's engine type that converts information to free energy. We pump heat from an isothermal environment by using the information about the thermal fluctuations of a Brownian particle and increase the particle's free energy.

Properties of the electrogenic activity of bacteriorhodopsin.

In this study, we analyzed the photoelectric current generated by bacteriorhodopsin adsorbed on a polymer film, "Lumirror" (Muneyuki et al. in FEBS Lett 427:109-114, 1998). We could examine the photoelectric current over a wide range of light intensity and pH values using the same membrane owing to the mechanical and chemical stability of the thin polymer film. We analyzed the photoelectric current by comparison with a simple equivalent electric circuit. Analysis of experimental results obtained at different light intensities suggested that the electromotive force of the bacteriorhodopsin was independent of light intensity. The pH dependence of the photoelectric current suggested that the bacteriorhodopsin could generate a maximum electromotive force at approximately pH 6.

Thermodynamic efficiency and mechanochemical coupling of F1-ATPase.

F(1)-ATPase is a nanosized biological energy transducer working as part of F(o)F(1)-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F(1)-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F(1)-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of F(o)F(1)-ATP synthase. We found that the maximum work performed by F(1)-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F(1)-ATPase.

Fabrication and placement of a ring structure of nanoparticles by a laser-induced micronanobubble on a gold surface.

We have developed a new fabrication method for a ring structure of assembled nanoparticles on a gold surface by the use of continuous Nd:YAG laser light. A micronanobubble on a gold surface, created by laser local heating, acts as a template for the formation of the ring structure. Both Marangoni convection flow and capillary flow around the micronanobubble are responsible for the driving force to assemble nanoparticles such as CdSe Q-dots into the ring structure from the solution. Because a single micronanobubble was generated by the Nd:YAG laser focusing point, the precise positioning of the ring structure was feasible directly on the gold surface, which makes it possible to fabricate various patterns of rings such as arrays and letters and even a double-ring structure without any photomasks or any templates.

Nonequilibrium energetics of a single F1-ATPase molecule.

Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method. We show that the sum of the heat flow through the probe's rotational degree of freedom and the work against an external load is almost equal to the free energy change per a single ATP hydrolysis under various conditions. This implies that F1-ATPase works at an efficiency of nearly 100% in a thermally fluctuating environment.

Observation of DNA pinning at laser focal point on Au surface and its application to single DNA nanowire and cross-wire formation.

We report a new technique for fabricating a single DNA nanowire at a desired position in a sequential manner using the micronanobubble generated by laser local heating at the Au/water interface. In our previous report, we found the reversible pull-in/shrinkage of one end immobilized DNA strands near a Nd:YAG laser focal point on an Au surface. In further experiments, the pinning of DNA strands in the stretched state was observed on the Au surface only when the bubble has touched the free end of DNA. This pinning phenomenon was observed even on the alkane thiol modified Au surface as self-assembled monolayers (SAMs) such as hexanethiol, mercaptohexanol, and hexadecanethiol. However, no pinning was observed on the bovine serum albumin (BSA) modified surface. Since optical tweezers can manipulate a DNA modified bead (radius=1.87 µm), the bead was firstly fixed on a solid surface by being compressed with the optical tweezers, and the pulling and pinning of DNA on the bead were achieved. As a consequence, the laser local heating on the Au surface enables us to control the number and position of the one end immobilized DNA strands as DNA nanowires.

Energy dissipation of a Brownian particle in a viscoelastic fluid.

We evaluate the energy dissipation rate of an optically driven Brownian particle in a polymer solution utilizing the generalized version of Harada and Sasa's equality [Phys. Rev. Lett. 95, 130602 (2005)] by Deutsch and Narayan [Phys. Rev. E 74, 026112 (2006)]. The irreversible work of a small system is estimated from readily obtainable quantities. By adopting the time-dependent memory function obtained by microrheology measurement, directly obtained works are in excellent agreement with those calculated from the generalized fluctuation dissipation theorem for nonequilibrium steady states. This result implies that the colloidal particle in a polymer solution can be described by the generalized Langevin equation.