Biology subjects

Iino, R.

Publications and source records attributed to Iino, R..

8 recordsLinked to original sources

bioRxiv2026

Artificial DNA-nano/microparticle motors: Factors governing speed, run-length, and unidirectionality revealed by geometry-based kinetic simulations

DNA-nano/microparticle motors are burnt-bridge Brownian ratchets (BBR) moving on an RNA-modified two-dimensional surface driven by Ribonuclease H (RNase H), and are one of the fastest artificial molecular motors. Interestingly, these motors show a maximum speed of [~]30 nm s-{superscript 1} irrespective of the particle size ranging from 100 to 5000 nm, whereas the run-length increases with the particle size. Here we performed geometry-based kinetic simulations of DNA-nano/microparticle motors with the sizes of 100, 500, 1000, and 5000 nm to identify the factors governing speed, run-length, and unidirectionality. The simulations reproduced the experiments quantitatively, and the speed remained constant while the run-length and the unidirectionality increased with the particle size. The constant speed was caused by a trade-off between the step size and the pause length, both of which increased with the particle size. In contrast, the run-length and the unidirectionality increased with the particle size because large particles had high multivalency which suppresses stochastic detachment of the motor, high RNA hydrolysis efficiency under the motor trajectory which realizes almost perfect BBR motion, and stepping direction highly biased to forward. For the smaller motors with 100, 500, and 1000 nm particles, the speed increased from 20 to 200 nm s-{superscript 1} by 10-fold increases in DNA/RNA hybridization, RNase H binding, and RNA hydrolysis rates (from 0.8 to 8.0, 7.2 to 72, and 3.0 to 30 s-{superscript 1}, respectively), even when considering the rotational diffusion of these particles. On the other hand, the speed for the largest motor with 5000 nm particle was limited to 100 nm s-{superscript 1}, because the time required for rolling motion ([~]0.3 s) became comparable to the pause length. Our results indicate that DNA-particle motors must possess a nanoscale body to achieve a speed exceeding 100 nm s-{superscript 1}. SignificanceAutonomous artificial molecular motors have a potential to power nano- and micron-scale actuators and devices, but their performances such as speed, run-length, and unidirectionality are inferior to natural motor proteins. Using geometry-based kinetic simulations, we quantitatively analyzed performance metrics of artificial DNA-nano/microparticle motors which autonomously move on RNA-modified two-dimensional surfaces by a burnt-bridge Brownian ratchet mechanism. Our study revealed the mechanism why their speed is almost independent of the particle size, while the run-length and unidirectionality increases with the particle size. We also identified how multivalent binding, mode of detachment, and rotational diffusion set fundamental limits of the speed, run-length, and unidirectionality. Our results provide a general design strategy for engineering high-performance artificial molecular motors.

biophysics↗

bioRxiv2025

Enhancing the imaging rate of high-speed atomic force microscopy using a combination of multiple techniques

Protein molecules, while functioning, undergo dynamic conformational changes and interactions with their partners, which can be directly observed by high-speed atomic force microscopy (HS-AFM). However, its imaging rate of approximately 10 frames per second (fps) has remained unchanged since its inception, despite the increasing demand for observing dynamic biomolecular processes that occur too fast to be captured at this time resolution. Here we report a combination of techniques that significantly enhances the imaging rate without disturbing the sample. HS-AFM imaging of the stator ring of F1-ATPase visualised two intermediate states, which could not be resolved with the previous HS-AFM system. The high-resolution molecular movies demonstrate how the catalytic subunits cooperate during the chemomechanical cycle.

biophysics↗

bioRxiv2025

A dual-domain chitinase mechanism enables marine bacteria tosense and localize sparse crystalline chitin in the ocean

Chitin is one of the most abundant biopolymers in the ocean, and its remineralisation underpins major pathways of marine carbon cycling. A central challenge for chitinolytic bacteria is to locate and remain associated with sparsely distributed crystalline chitin particles. Here, we show that the GH18 chitinase VpChi1 from Vibrio parahaemolyticus possesses a dual-domain architecture that enhances particle encounter and retention. Full-length VpChi1 displayed much higher catalytic efficiency than a CBD-truncated mutant at low chitin concentrations, whereas substrate inhibition occurred at higher concentrations. Single-molecule fluorescence imaging demonstrated that the additional C-terminal chitin-binding domain (CBD) enables selective binding to the hydrophobic surface of crystalline chitin and substantially prolongs enzyme residence time. Kinetic simulations using experimentally determined dissociation rates showed that this extended residence arises from CBD-assisted rebinding of the catalytic domain. High-speed atomic force microscopy confirmed that the CBD does not alter stepping velocity or travel distance along individual crystals, indicating that substrate inhibition likely results from CBD engagement with neighbouring particles rather than impaired motility. Screening of seawater and marine sediments across multiple depths identified Vibrio and Pseudoalteromonas species as dominant chitin degraders, many encoding GH18 chitinases with dual CBDs, whereas terrestrial bacteria generally lacked this architecture. We propose that this two-domain configuration confers an ecological advantage by increasing enzyme retention on particulate chitin and enabling sustained production of chitobiose, a potent chemotactic signal for marine bacteria. These findings reveal a mechanistic basis for how bacteria sense and exploit particulate chitin and highlight an adaptive strategy for resource acquisition in the ocean.

molecular biology↗

bioRxiv2025

Neck-region-microtubule interactions direct counterclockwise stepping of kinesin-1

The "neck" region of kinesin is a structurally conserved element critical for force generation, stepping directionality, and cargo transport along microtubules, yet its atomic-scale structure in a functional context remains unresolved. Here, we employ all-atom replica exchange molecular dynamics simulations to resolve a high-confidence conformation of the neck region in dimeric human kinesin-1 bound to a realistic microtubule lattice, and use this structure to simulate kinesins initial stepping motion. Our simulations reveal that the neck coiled-coil is oriented perpendicular to the microtubules axis and positioned near its surface--a conformation consistent with earlier proposals but lacking high-resolution validation. Importantly, simulations indicate that neck-microtubule interactions bias the stepping trajectory, directing the rear kinesin head to overtake the front head from the right (counterclockwise stepping). These findings establish a mechanism by which neck-microtubule interactions govern directional bias in kinesins initial step, offering new insight into the molecular basis of its motility.

biophysics↗

bioRxiv2024

ATP synthesis of Enterococcus hirae V-ATPase driven by sodium motive force

V-ATPases generally function as ion pumps driven by ATP hydrolysis in the cell, but their capability of ATP synthesis remains largely unexplored. Here we show ATP synthesis of Na+-transporting Enterococcus hirae V-ATPase (EhVoV1) driven by electrochemical potential gradient of Na+ across the membrane (sodium motive force, smf). We reconstituted EhVoV1 into liposome and performed a luciferin/luciferase-based assay to analyze ATP synthesis quantitatively. Our result demonstrates that EhVoV1 synthesizes ATP with a rate of 4.7 s-1 under high smf (269.3 mV). The Michaelis constants for ADP (21 {micro}M) and inorganic phosphate (2.1 mM) in ATP synthesis reaction were comparable to those for ATP synthases, suggesting similar substrate affinities among rotary ATPases regardless of their physiological functions. Both components of smf, Na+ concentration gradient across the membrane ({Delta}pNa) and membrane potential ({Delta}{psi}), contributed to ATP synthesis, with {Delta}pNa showing a slightly larger impact. At the equilibrium points where smf and Gibbs free energy of ATP synthesis are balanced, EhVoV1 showed reversible reactions between ATP synthesis and hydrolysis. The obtained Na+/ATP ratio (3.2 {+/-} 0.4) closely matched the value expected from the structural symmetry ratio between EhVo and EhV1 (10/3 = 3.3), indicating tight coupling between ATP synthesis/hydrolysis and Na+ transport. These results reveal inherent functional reversibility of EhVoV1. We propose that physiological function of EhVoV1 in vivo is determined by relatively small smf against large Gibbs free energy of ATP synthesis, in addition to the absence of inhibitory mechanisms of ATP hydrolysis which are known for ATP synthases.

biophysics↗

bioRxiv2024

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 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.

biophysics↗

bioRxiv2020

De Novo Design of Allosteric Control into Rotary Motor V1-ATPase by Restoring Lost Function

Protein complexes exert various functions through allosterically controlled cooperative work. De novo design of allosteric control into protein complexes provides understanding of their working principles and potential tools for synthetic biology. Here, we hypothesized that an allosteric control can be created by restoring lost functions of pseudo-enzymes contained as subunits in protein complexes. This was demonstrated by computationally de novo designing ATP binding ability of the pseudo-enzyme subunits in a rotary molecular motor, V1-ATPase. Single molecule experiments with solved crystal structures revealed that the designed V1 is allosterically accelerated than the wild-type by the ATP binding to the created allosteric site and the rate is tunable by modulating the binding affinity. This work opened up an avenue for programming allosteric control into proteins exhibiting concerted functions.

biophysics↗

bioRxiv2019

Dynamic free energy landscape of a linear motor chitinase from single-particle tracking trajectories

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 singlemolecule 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 singlemolecule 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.

biophysics↗