Visualizing Single V-ATPase Rotation Using Janus Nanoparticles.

Understanding the function of rotary molecular motors, such as rotary ATPases, relies on our ability to visualize single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from the translational motion of NPs. Here, we report an approach using "two-faced" Janus NPs to directly image the rotation of a single V-ATPase from , an ATP-driven rotary ion pump.

Na-V-ATPase inhibitor curbs VRE growth and unveils Na pathway structure.

Vancomycin-resistant Enterococcus faecium (VRE) is a major cause of nosocomial infections, particularly endocarditis and sepsis. With the diminishing effectiveness of antibiotics against VRE, new antimicrobial agents are urgently needed. Our previous research demonstrated the crucial role of Na-transporting V-ATPase in Enterococcus hirae for growth under alkaline conditions.

Visualizing Single V-ATPase Rotation Using Janus Nanoparticles.

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 , an ATP-driven rotary ion pump.

Changes in the expression of mexB, mexY, and oprD in clinical Pseudomonas aeruginosa isolates.

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.

Sub-Tip-Radius Near-Field Interactions in Nano-FTIR Vibrational Spectroscopy on Single Proteins.

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.

Six states of Enterococcus hirae V-type ATPase reveals non-uniform rotor rotation during turnover.

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 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.

Design of allosteric sites into rotary motor V-ATPase by restoring lost function of pseudo-active sites.

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.

Direct observation of stepping rotation of V-ATPase reveals rigid component in coupling between V and V motors.

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 and V, and V-ATPase (EhVV) actively transports Na in V (EhV) by using torque generated by ATP hydrolysis in V (EhV). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhVV 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.

Linear-Zero Mode Waveguides for Single-Molecule Fluorescence Observation of Nucleotides in Kinesin-Microtubule Motility Assay.

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.

Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy.

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.

Crystalline chitin hydrolase is a burnt-bridge Brownian motor.

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 (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose.

Combined Approach to Engineer a Highly Active Mutant of Processive Chitinase Hydrolyzing Crystalline Chitin.

chitinase A (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 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.

Single Cell Array Enclosed with a Photodegradable Hydrogel in Microwells for Image-Based Cell Classification and Selective Photorelease of Cells.

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.

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

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 (CfCel6B) and cellobiohydrolase II from the fungus (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity.

Chemical-State-Dependent Free Energy Profile from Single-Molecule Trajectories of Biomolecular Motors: Application to Processive Chitinase.

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.

Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking.

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.

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from .

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 (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 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.

Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of V-ATPase.

V-ATPase (V), 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 from the pathogen (EhV). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps.

Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array.

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 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.

Publisher Correction: Dynamic structural states of ClpB involved in its disaggregation function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies.

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.

Single-Nanoparticle Tracking with Angstrom Localization Precision and Microsecond Time Resolution.

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.

Visualization of Functional Structure and Kinetic Dynamics of Cellulases.

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.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

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.