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Understanding the function of rotary molecular motors, such as the rotary ATPases, relies on our ability to visualize the single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from translational motion of NPs. Here, we report an approach using ″two-faced″ Janus NPs to directly image the rotation of single V-ATPase from Enterococcus hirae , an ATP-driven rotary ion pump. By employing a 500-nm silica/gold Janus NP, we exploit its asymmetric optical contrast - silica core with a gold cap on one hemisphere - to achieve precise imaging of the unidirectional counter-clockwise rotation of single V-ATPase motors immobilized on surfaces. Despite the added viscous load from the relatively large Janus NP probe, our approach provides accurate torque measurements of single V-ATPase. This study underscores the advantages of Janus NPs over conventional probes, establishing them as powerful tools for single-molecule analysis of rotary molecular motors.

Tip-enhanced vibrational spectroscopy has advanced to routinely attain nanoscale spatial resolution, with tip-enhanced Raman spectroscopy even achieving atomic-scale and submolecular sensitivity. Tip-enhanced infrared spectroscopy techniques, such as nano-FTIR and AFM-IR spectroscopy, have also enabled the nanoscale chemical analysis of molecular monolayers, inorganic nanoparticles, and protein complexes. However, fundamental limits of infrared nanospectroscopy in terms of spatial resolution and sensitivity have remained elusive, calling for a quantitative understanding of the near-field interactions in infrared nanocavities. Here, we demonstrate the application of nano-FTIR spectroscopy to probe the amide-I vibration of a single protein consisting of ∼500 amino acid residues. Detection with higher tip tapping demodulation harmonics up to the seventh order leads to pronounced enhancement in the peak amplitude of the vibrational resonance, originating from sub-tip-radius geometrical effects beyond dipole approximations. This quantitative characterization of single-nanometer near-field interactions opens the path toward employing infrared vibrational spectroscopy at the subnanoscale and single-molecule levels.

Changes in expression levels of drug efflux pump genes, mexB and mexY, and porin gene oprD in Pseudomonas aeruginosa were investigated in this study. Fifty-five multidrug-resistant P. aeruginosa (MDRP) strains were compared with 26 drug-sensitive strains and 21 strains resistant to a single antibiotic. The effect of the efflux inhibitor Phe-Arg-β-naphthylamide on drug susceptibility was determined, and gene expression was quantified using real-time quantitative real-time reverse transcription polymerase chain reaction. In addition, the levels of metallo-β-lactamase (MBL) and 6'-N-aminoglycoside acetyltransferase [AAC(6')-Iae] were investigated. Efflux pump inhibitor treatment increased the sensitivity to ciprofloxacin, aztreonam, and imipenem in 71%, 73%, and 29% of MDRPs, respectively. MBL and AAC(6')-Iae were detected in 38 (69%) and 34 (62%) MDRP strains, respectively. Meanwhile, 76% of MDRP strains exhibited more than 8-fold higher mexY expression than the reference strain PAO1. Furthermore, 69% of MDRP strains expressed oprD at levels less than 0.01-fold of those in PAO1. These findings indicated that efflux pump inhibitors in combination with ciprofloxacin or aztreonam might aid in treating MDRP infections.

Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. Here we describe an approach to create artificial allosteric sites in protein complexes. Certain protein complexes contain subunits with pseudo-active sites, which are believed to have lost functions during evolution. Our hypothesis is that allosteric sites in such protein complexes can be created by restoring the lost functions of pseudo-active sites. We used computational design to restore the lost ATP-binding ability of the pseudo-active site in the B subunit of a rotary molecular motor, V 1 -ATPase. Single-molecule experiments with X-ray crystallography analyses revealed that binding of ATP to the designed allosteric site boosts this V 1 's activity compared with the wild-type, and the rotation rate can be tuned by modulating ATP's binding affinity. Pseudo-active sites are widespread in nature, and our approach shows promise as a means of programming allosteric control over concerted functions of protein complexes., (© 2023. The Author(s).)

The vacuolar-type ATPase from Enterococcus hirae (EhV-ATPase) is a thus-far unique adaptation of V-ATPases, as it performs Na + transport and demonstrates an off-axis rotor assembly. Recent single molecule studies of the isolated V 1 domain have indicated that there are subpauses within the three major states of the pseudo three-fold symmetric rotary enzyme. However, there was no structural evidence for these. Herein we activate the EhV-ATPase complex with ATP and identified multiple structures consisting of a total of six states of this complex by using cryo-electron microscopy. The orientations of the rotor complex during turnover, especially in the intermediates, are not as perfectly uniform as expected. The densities in the nucleotide binding pockets in the V 1 domain indicate the different catalytic conditions for the six conformations. The off-axis rotor and its' interactions with the stator a-subunit during rotation suggests that this non-uniform rotor rotation is performed through the entire complex., (© 2023. The Author(s).)

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, V o and V 1 , and Enterococcus hirae V-ATPase (EhV o V 1 ) actively transports Na + in V o (EhV o ) by using torque generated by ATP hydrolysis in V 1 (EhV 1 ). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhV o V 1 wild-type, aE634A, and BR350K mutants under various Na + and ATP concentrations ([Na + ] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na + ] was low and [ATP] was high, under the condition that only Na + binding to EhV o is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhV o rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na + ], confirming that Na + binding triggers the steps. When both [ATP] and [Na + ] were low, under the condition that both Na + and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhV o and EhV 1 , respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhV o and EhV 1 do not share pausing positions, Na + and ATP bindings occur at different angles, and the coupling between EhV o and EhV 1 has a rigid component.

Single-molecule fluorescence microscopy is a key tool to investigate the chemo-mechanical coupling of microtubule-associated motor proteins, such as kinesin. However, a major limitation of the implementation of single-molecule observation is the concentration of fluorescently labeled molecules. For example, in total internal reflection fluorescence microscopy, the available concentration is of the order of 10 nM. This concentration is much lower than the concentration of adenosine triphosphate (ATP) in vivo, hindering the single-molecule observation of fluorescently labeled ATP hydrolyzed by motor proteins under the physiologically relevant conditions. Here, we provide a method for the use of single-molecule fluorescence microscopy in the presence of ~500 nM of fluorescently labeled ATP. To achieve this, a device equipped with nano-slits is used to confine excitation light into its slits as an expansion of zero-mode waveguides (ZMWs). Conventional ZMWs equip apertures with a diameter smaller than the wavelength of light to suppress background noise from the labeled molecules diffusing outside of the apertures. While they are not compatible with filamentous objects, our linear-ZMWs enable the usage of filamentous objects, such as microtubules. An experiment using linear-ZMWs demonstrated the successful exploration of the interaction between kinesin and ATP using single-molecule fluorescence microscopy., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm-1, which did not appear in the reaction product, N,N'-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm-1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N'-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nakamura et al.)

Serratia marcescens chitinase A ( Sm ChiA) processively hydrolyzes recalcitrant biomass crystalline chitin under mild conditions. Here, we combined multiple sequence alignment, site-saturation mutagenesis, and automated protein purification and activity measurement with liquid-handling robot to reduce the number of mutation trials and shorten the screening time for hydrolytic activity improvement of Sm ChiA. The amino acid residues, which are not conserved in the alignment and are close to the aromatic residues along the substrate-binding sites in the crystal structure, were selected for site-saturation mutagenesis. Using the previously identified highly active F232W/F396W mutant as a template, we identified the F232W/F396W/S538V mutant, which shows further improved hydrolytic activity just by trying eight different sites. Importantly, valine was not found in the multiple sequence alignment at Ser538 site of Sm ChiA. Our combined approach allows engineering of highly active enzyme mutants, which cannot be identified only by the introduction of predominant amino acid residues in the multiple sequence alignment., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

Single cell arrays provide an accurate classification of analyte cells through an image-based analysis of cellular phenotypes. Light-guided cell retrieval from a single cell array is a promising approach for the rapid and simple sorting of difficult to distinguish cells. In this study, we developed a single cell array enclosed with a photodegradable hydrogel in microwells to enable both comprehensive image-based single cell analysis and light-guided cell retrieval. In this system, individual cells became trapped in the microwells together with the photodegradable hydrogel at a high cell density on a chip regardless of cell type, adhesiveness, and motility. Fluorescence-stained model cells and vaccinated dendritic cells were identified by microscopic imaging and then selectively released through the light-induced degradation of the cell-embedding hydrogels. The target cells were selectively retrieved with a purity of >95% from the cell mixture through rapid photorelease, and the retrieved cells were confirmed to grow normally. Our results provide proof-of-principle that the photoresponsive microwell array serves as a versatile tool for image-based cell sorting in cellular researches and the manufacturing processes of high-performance cells.

The mechanism of biomolecular motors has been elucidated using single-molecule experiments for visualizing motor motion. However, it remains elusive that how changes in the chemical state during the catalytic cycle of motors lead to unidirectional motions. In this study, we use single-molecule trajectories to estimate an underlying diffusion model with chemical-state-dependent free energy profile. To consider nonequilibrium trajectories driven by the chemical energy consumed by biomolecular motors, we develop a novel framework based on a hidden Markov model, wherein switching among multiple energy profiles occurs reflecting the chemical state changes in motors. The method is tested using simulation trajectories and applied to single-molecule trajectories of processive chitinase, a linear motor that is driven by the hydrolysis energy of a single chitin chain. The chemical-state-dependent free energy profile underlying the burnt-bridge Brownian ratchet mechanism of processive chitinase is determined. The novel framework allows us to connect the chemical state changes to the unidirectional motion of biomolecular motors.

Motor proteins are essential units of life and are well-designed nanomachines working under thermal fluctuations. These proteins control moving direction by consuming chemical energy or by dissipating electrochemical potentials. Chitinase A from bacterium Serratia marcescens (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose. Recently, we directly observed the stepping motions of SmChiA labeled with a gold nanoparticle by dark-field scattering imaging to investigate the moving mechanism. Time constants analysis revealed that SmChiA moves back and forth along the chain freely, because forward and backward states have a similar free energy level. The similar probabilities of forward-step events (83.5%=69.3%+14.2%) from distributions of step sizes and chain-hydrolysis (86.3%=(1/2.9)/(1/2.9+1/18.3)×100) calculated from the ratios of time constants of hydrolysis and the backward step indicated that SmChiA moves forward as a result of shortening of the chain by a chitobiose unit, which stabilizes the backward state. Furthermore, X-ray crystal structures of sliding intermediate and molecular dynamics simulations showed that SmChiA slides forward and backward under thermal fluctuation without large conformational changes of the protein. Our results demonstrate that SmChiA is a burnt-bridge Brownian ratchet motor., (2020 THE BIOPHYSICAL SOCIETY OF JAPAN.)

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens ( Sm ChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of Sm ChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of Sm ChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 Sm ChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for Sm ChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of Sm ChiA mutants., (© 2020 Visootsat et al.)

Background: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy., Methods: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever., Results: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated., Conclusions: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research., General Significance: We achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 μs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between αβ-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed.

V 1 -ATPase (V 1 ), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis-derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V 1 from the pathogen Enterococcus hirae (EhV 1 ). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 10 7 m -1 s -1 At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, -80 and -40° backward steps were observed. The time constants of the pauses before -80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV 1 , including substeps largely different from those of F 1 -ATPases., (© 2019 Iida et al.)

We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm 2 to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution-predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 μM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

Gold nanoparticles (AuNPs) have been used as a contrast agent for optical imaging of various single biomolecules. Because AuNPs have high scattering efficiency without photobleaching, biomolecular dynamics have been observed with nanometer localization precision and sub-millisecond time resolution. To understand the working principle of biomolecular motors in greater detail, further improvement of the localization precision and time resolution is necessary. Here, we investigated the lower limit of localization precision achievable with AuNPs and the fundamental law, which determines the localization precision. We first used objective-lens-type total internal reflection dark-field microscopy to obtain a scattering signal from an isolated AuNP. The localization precision was inversely proportional to the square root of the photon number, which is consistent with theoretical estimation. The lower limit of precision for a 40 nm AuNP was ∼0.3 nm with 1 ms time resolution and was restricted by detector saturation. To achieve higher localization precision, we designed and constructed an annular illumination total internal reflection dark-field microscopy system with an axicon lens, which can illuminate the AuNPs at high laser intensity without damaging the objective lens. In addition, we used high image magnification to avoid detector saturation. Consequently, we achieved 1.3 Å localization precision for 40 nm AuNPs and 1.9 Å localization precision for 30 nm AuNPs at 1 ms time resolution. Furthermore, even at 33 μs time resolution, localization precisions at 5.4 Å for 40 nm AuNPs and at 1.7 nm for 30 nm AuNPs were achieved. We then observed motion of head of kinesin-1 labeled with AuNP at microsecond time resolution. Transition cycles of bound/unbound states and tethered diffusion of unbound head during stepping motion on microtubule were clearly captured with higher time resolution or smaller AuNP than those used in previous studies, indicating applicability to single-molecule imaging of biomolecular motors., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

EhV-ATPase is an ATP-driven Na + pump in the eubacteria Enterococcus hirae (Eh). Here, we present the first entire structure of detergent-solubilized EhV-ATPase by single-particle cryo-electron microscopy (cryo-EM) using Zernike phase plate. The cryo-EM map dominantly showed one of three catalytic conformations in this rotary enzyme. To further stabilize the originally heterogeneous structure caused by the ATP hydrolysis states of the V 1 -ATPases, a peptide epitope tag system was adopted, in which the inserted peptide epitope sequence interfered with rotation of the central rotor by binding the Fab. As a result, the map unexpectedly showed another catalytic conformation of EhV-ATPase. Interestingly, these two conformations identified with and without Fab conversely coincided with those of the minor state 2 and the major state 1 of Thermus thermophilus V/A-ATPase, respectively. The most prominent feature in EhV-ATPase was the off-axis rotor, where the cytoplasmic V 1 domain was connected to the transmembrane V o domain through the off-axis central rotor. Furthermore, compared to the structure of ATP synthases, the larger size of the interface between the transmembrane a-subunit and c-ring of EhV-ATPase would be more advantageous for active ion pumping.

Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s -1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

We evaluated a method for protein engineering using plasmid-based one-pot saturation mutagenesis and robot-based automated screening. When the biases in nucleotides and amino acids were assessed for a loss-of-function point mutation in green fluorescent protein, the ratios of gain-of-function mutants were not significantly different from the expected values for the primers among the three different suppliers. However, deep sequencing analysis revealed that the ratios of nucleotides in the primers were highly biased among the suppliers. Biases for NNB were less severe than for NNN. We applied this method to screen a fusion protein of two chitinases, ChiA and ChiB (ChiAB). Three NNB codons as well as tyrosine and serine (X 1 YSX 2 X 3 ) were inserted to modify the surface structure of ChiAB. We observed significant amino acid bias at the X 3 position in water-soluble, active ChiAB-X 1 YSX 2 X 3 mutants. Examination of the crystal structure of one active mutant, ChiAB-FYSFV, revealed that the X 3 residue plays an important role in structure stabilization., Competing Interests: The authors declare no competing financial interest.

The ATP-dependent bacterial protein disaggregation machine, ClpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable ClpB to disentangle protein aggregates.

Background: Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration., Scope of Review: We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems., Major Conclusions: Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines., General Significance: Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (k on ), translational movement (k tr ), and dissociation (k off ) with single-molecule fluorescence imaging. The k on for a single chitin microfibril was 2.1 × 10 9 M -1 μm -1 s -1 . The k off showed two components, k (3.2 s -1 , 78%) and k (0.38 s -1 , 22%), corresponding to bindings to different crystal surfaces. From the k on , k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10 -9 M μm and 8.1 × 10 -10 M μm, respectively. The k tr was 52.5 nm s -1 , and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s -1 ) calculated from k tr and biochemically determined low k cat (2.6 s -1 ) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s -1 × 0.048 = 2.5 s -1 ). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem. Chem. Phys., 2018, DOI: .

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

Cellulose is the most abundant carbohydrate on earth and hydrolyzed by cellulases in nature. During catalysis, cellulase transfers protons to and from the oxygen atoms of the glycosidic bond and a water molecule. Since cellulose is an insoluble polymer, some kinds of cellulases, with high activity toward crystalline cellulose, move on the crystal surface with continuous hydrolysis of the molecular chain. In addition, binding and dissociation on/from the crystal surface are also important elementary steps of the reaction cycle. Recently, these interesting features of cellulases can be directly analyzed, due to the development of visualization techniques. In this chapter, we introduce (1) visualization of the protonation state of the catalytic residue by neutron crystallography, (2) visualization of processive movement on the crystal surface by high-speed atomic force microscopy , and (3) visualization of binding and dissociation events by single-molecule fluorescence microscopy.

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose I α The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

F 1 - and V 1 -ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F 1 or of V 1 and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F 1 or V 1 These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F 1 and V 1 The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F 1 -FliJ chimera generates only 10% of WT F 1 , the V 1 -FliJ chimera generates torque comparable to that of V 1 with the native axle protein that is structurally more similar to FliJ than the native rotor of F 1 This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F 1 ., Competing Interests: The authors declare no conflict of interest.

The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM) device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller-Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.

A directly accessible femtoliter droplet array as a platform for single-cell detection and collection of persister bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single cells, long-term culture and observation of single cells in droplets, and collection of identified persisters from single droplets are described in detail.

Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.

In ion-transporting rotary ATPases, the mechanical rotation of inner rotor subunits against other stator subunits in the complex mediates conversion of chemical free energy from ATP hydrolysis into electrochemical potential by pumping ions across the cell membrane. To fully understand the rotational mechanism of energy conversion, it is essential to analyze a target sample by multiple advanced methods that differ in spatiotemporal resolutions and sample environments. Here, we describe such a strategy applied to the water-soluble V1 moiety of Enterococcus hirae V-ATPase; this strategy involves integration of crystal structure studies and single-molecule analysis of rotary dynamics and torque generation. In addition, we describe our current model of the chemo-mechanical coupling scheme obtained by this approach, as well as future prospects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of ∼1° for the azimuthal angle at a 10 μs temporal resolution, the defocused image analysis showed a superior angle accuracy of ∼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of ∼1°, by fitting with simulated images. By taking advantage of the defocused image method's full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F1-ATPase, was measured with 3.3 μs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines.

A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F1-ATPase (F1), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F1 in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH2 unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F1. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high "chemical" resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics.

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Single-molecule imaging and manipulation with optical microscopy have become essential methods for studying biomolecular machines; however, only few efforts have been directed towards synthetic molecular machines. Single-molecule optical microscopy was now applied to a synthetic molecular rotor, a double-decker porphyrin (DD). By attaching a magnetic bead (ca. 200 nm) to the DD, its rotational dynamics were captured with a time resolution of 0.5 ms. DD showed rotational diffusion with 90° steps, which is consistent with its four-fold structural symmetry. Kinetic analysis revealed the first-order kinetics of the 90° step with a rate constant of 2.8 s(-1). The barrier height of the rotational potential was estimated to be greater than 7.4 kJ mol(-1) at 298 K. The DD was also forcibly rotated with magnetic tweezers, and again, four stable pausing angles that are separated by 90° were observed. These results demonstrate the potency of single-molecule optical microscopy for the elucidation of elementary properties of synthetic molecular machines., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the high-resolution molecular structure of Enterococcus hirae V1-ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemomechanical coupling scheme., (© 2014 International Union of Biochemistry and Molecular Biology.)

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

Single-cell analysis is a powerful method to assess the heterogeneity among individual cells, enabling the identification of very rare cells with properties that differ from those of the majority. In this Methods Article, we describe the use of a large-scale femtoliter droplet array to enclose, isolate, and analyze individual bacterial cells. As a first example, we describe the single-cell detection of drug-tolerant persisters of Pseudomonas aeruginosa treated with the antibiotic carbenicillin. As a second example, this method was applied to the single-cell evaluation of drug efflux activity, which causes acquired antibiotic resistance of bacteria. The activity of the MexAB-OprM multidrug efflux pump system from Pseudomonas aeruginosa was expressed in Escherichia coli and the effect of an inhibitor D13-9001 were assessed at the single cell level.

High-speed atomic force microscopy (HS-AFM) and total internal reflection fluorescence microscopy (TIRFM) have mutually complementary capabilities. Here, we report techniques to combine these microscopy systems so that both microscopy capabilities can be simultaneously used in the full extent. To combine the two systems, we have developed a tip-scan type HS-AFM instrument equipped with a device by which the laser beam from the optical lever detector can track the cantilever motion in the X- and Y-directions. This stand-alone HS-AFM system is mounted on an inverted optical microscope stage with a wide-area scanner. The capability of this combined system is demonstrated by simultaneous HS-AFM∕TIRFM imaging of chitinase A moving on a chitin crystalline fiber and myosin V walking on an actin filament.

Ring-shaped nucleoside triphosphatases (ring NTPases) are biological molecular machines powered by energy from NTP hydrolysis and are responsible for various cellular activities. These ring NTPases translocate their substrates or rotate their own subunits to/in the hole of the ring. Coordination and cooperativity among subunits in the oligomer ring is a topic of debate focused on understanding the operation mechanism of these protein machines. With the help of X-ray crystallographic structural analysis and optical microscopic single-molecules studies, distinct models, including stochastic, concerted, and rotary catalysis have been proposed. Here, we discuss these models and introduce high-speed atomic force microscopy as a new potent tool for verification of the model, with our recent example of the rotary catalysis of the stator ring of F1-adenosine triphosphatase., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

F(o) F(1) -Adenosine triphosphate (ATP) synthase, a complex of two rotary motor proteins, reversibly converts the electrochemical potential of protons across the cell membrane into phosphate transfer potential of ATP to provide the energy currency of the cell. The water-soluble motor is F(1) -ATPase, which possesses ATP synthesis/hydrolysis catalytic sites. Isolated F(1) hydrolyses ATP to rotate the rotary shaft against the stator ring. The membrane-embedded motor is F(o) , which is driven by proton flow down the proton electrochemical potential. In the F(o) F(1) complex, the direction of mechanical rotation, the chemical reaction, and the proton transport are determined by the relative amplitudes between the Gibbs free energy of the ATP hydrolysis reaction and the electrochemical potential of protons across the membrane. Therefore, F(o) F(1) -ATP synthase is a highly efficient molecular device in which the chemical, mechanical, and potential energies are tightly and reversibly converted. In this critical review, we summarize our latest knowledge about the operation mechanism of this sophisticated nanomachine, revealed by its structure and dynamics., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)