Biomolecular Motors Research Articles

Surface Passivation of Norland Optical Adhesive Improves the Guiding Efficiency of Gliding Microtubules in Microfluidic Devices.

The microtubule-kinesin biomolecular motor system, which is vital for cellular function, holds significant promise for nanotechnological applications. In vitro gliding assays have demonstrated the ability to transport microcargo by propelling microtubules across kinesin-coated surfaces. However, the uncontrolled directional motion of microtubules has posed significant challenges, limiting the system's application for precise cargo delivery. Microfluidic devices provide a means to direct microtubule movement through their geometric features. Norland Optical Adhesive (NOA) is valued for its mold-free application in microfluidic device fabrication; however, microtubules often climb up channel walls, limiting controlled movement. In this study, a surface passivation method for NOA is introduced, using polyethylene glycol via a thiol-ene click reaction. This technique significantly improved the directional control and concentration of microtubules within NOA microchannels. This approach presents new possibilities for the precise application of biomolecular motors in nanotechnology, enabling advancements in the design of microfluidic systems for complex biomolecular manipulations.

Exerting pulling forces in fluids by directional disassembly of microcrystalline fibres

Biomolecular polymerization motors are biochemical systems that use supramolecular (de-)polymerization to convert chemical potential into useful mechanical work. With the intent to explore new chemomechanical transduction strategies, here we show a synthetic molecular system that can generate forces via the controlled disassembly of self-organized molecules in a crystal lattice, as they are freely suspended in a fluid. An amphiphilic monomer self-assembles into rigid, high-aspect-ratio microcrystalline fibres. The assembly process is regulated by a coumarin-based pH switching motif. The microfibre crystal morphology determines the monomer reactivity at the interface, resulting in anisotropic etching. This effect exerts a directional pulling force on microscopic beads adsorbed on the crystal surface through weak multivalent interactions. We use optical-tweezers-based force spectroscopy to extract mechanistic insights into this process, quantifying a stall force of 2.3 pN (±0.1 pN) exerted by the ratcheting mechanism produced by the disassembly of the microfibres.

Rotary FoF1-ATP Synthase-Driven Flasklike Pentosan Colloidal Motors with ATP Synthesis and Storage.

We report the hierarchical assembly of a chloroplast-derived rotary FoF1-ATPase motor-propelled flasklike pentosan colloidal motor (FPCM) with the ability of the synthesis, storage, and triggered release of biological energy currency ATP. These streamlined and submicrometer-sized hollow flasklike pentosan colloidal motors are prepared by combining a soft-template-based hydrothermal polymerization with a vacuum infusion of chloroplast-derived proteoliposomes containing rotary FoF1-ATPase motors. The generation of proton motive force across the proteoliposomes by injecting an acidic buffer solution promotes the rotation of FoF1-ATPase motors to drive the self-propelled motion of FPCMs, accompanying the inner ATP synthesis and storage. These rotary FoF1-ATPase motor-powered FPCMs exhibit a chemotactic behavior by migrating from their neck opening to their round bottom along a proton gradient of the external environment (negative chemotaxis). Such rotary biomolecular motor-driven flasklike pentosan colloidal motors with ATP synthesis and on-demand release make them promising candidates for engineering novel intelligent nanocarriers to actively regulate cellular metabolism.

An FOF1-ATPase motor-embedded chromatophore as a nanorobot for overcoming biological barriers and targeting acidic tumor sites

Despite the booming progress of anticancer nanomedicines in the past two decades, precise tumor-targetability and sufficient tumor-accumulation are less successful and still require further research. To tackle this challenge, herein we present a biomolecular motor (FOF1-ATPase)-embedded chromatophore as nanorobot to efficiently overcome biological barriers, and thoroughly investigate its chemotactic motility, tumor-accumulation ability and endocytosis. Chromatophores embedded with FOF1-ATPase motors were firstly extracted from Thermus thermophilus, then their properties were fully characterized. Specifically, two microfluidic platforms (laminar flow microchip and tumor microenvironment (TME) microchip) were designed and developed to fully investigate the motility, tumor-accumulation ability and endocytosis of the chromatophore nanorobot (CN). The results from the laminar flow microchip indicated that the obtained CN possessed the strongly positive chemotaxis towards protons. And the TME microchip experiments verified that the CN had a desirable tumor-accumulation ability. Cellular uptake experiments demonstrated that the CN efficiently promoted the endocytosis of the fluorescence DiO into the HT-29 cells. And the in vivo studies revealed that the intravenously administered CN exhibited vigorous tumor-targetability and accumulation ability as well as highly efficient antitumor efficacy. All the results suggested that FOF1-ATPase motors-embedded CN could be promising nanomachines with powerful self-propulsion for overcoming physiological barriers and tumor-targeted drug delivery. Statement of significanceIn this study, we demonstrated that FOF1-ATPase-embedded chromatophore nanorobots exhibit a strong proton chemotaxis, which not only plays a key role in tumor-targetability and accumulation, but also promotes tumor tissue penetration and internalization. The results of in vitro and in vivo studies indicated that drug-loaded chromatophore nanorobots are capable to simultaneously accomplish tumor-targeting, accumulation, penetration and internalization for enhanced tumor therapy. Our study provides a fundamental basis for further study on FOF1-ATPase-embedded chromatophore as tumor-targeting drug delivery systems that have promising clinical applications. It offers a new and more efficient delivery vehicle for cancer related therapeutics.

Unlocking the potential of information flow: Maximizing free-energy transduction in a model of an autonomous rotary molecular motor.

Molecular motors fulfill critical functions within all living beings. Understanding their underlying working principles is therefore of great interest. Here we develop a simple model inspired by the two-component biomolecular motor F_{o}-F_{1} ATP synthase. We analyze its energetics and characterize information flows between the machine's components. At maximum output power we find that information transduction plays a minor role for free-energy transduction. However, when the two components are coupled to different environments (e.g., when in contact with heat baths at different temperatures), we show that information flow becomes a resource worth exploiting to maximize free-energy transduction. Our findings suggest that real-world powerful and efficient information engines could be found in machines whose components are subjected to fluctuations of different strength, since in this situation the benefit gained from using information for work extraction can outweigh the costs of information generation.

Contractile and Tensile Measurement of Molecular Artificial Muscles for Biohybrid Robotics.

A printable artificial muscle assembled from biomolecular motors, which we have recently developed, showed great potential in overcoming the design limitations of conventional biohybrid robots as a new bio-actuator. Characterizing its contractility for extending its applicability is important. However, conventional measurement methods are composed of complex operations with poor reproducibility, flexibility, and real-time responsiveness. This study presents a new method for measuring the contractile force generated by artificial muscles. A measurement system was constructed, wherein artificial muscles were patterned by UV laser scanning in an oil-sealed microchamber, and the contractile force was measured in real time using a microforce sensor extended by a 3D-printed microcantilever. The measurement accuracy of the sensor was ensured through calibration and correction. For demonstration purposes, a series of contractile measurements were carried out using the proposed system. The relationship between contractile force and the dimensions of the activation space of the artificial muscles, as well as the tensile properties of the contracted muscle chain were evaluated. The results will help characterize the contractile properties of the artificial muscle and lay the foundations for its further application in biohybrid robotics.

(Invited) Integration of Functional Nanostructures and Nanoparticles into Micro- and Nanoelectronic Components and Systems

In recent decades, there has been a significant evolution of microelectronic and semiconductor technologies towards the nanoscale. Both bottom-up and top-down integration concepts of functional nanostructures and nanoparticles into micro- and nanofabricated electronic components have become critical issues that decisively affect the performance of smart electronic systems in various application areas such as environmental sensing and energy harvesting.Functional nanostructures and nanoparticles of different shapes, sizes, morphologies, and materials are now used in various technical and industrial applications, medicine, pharmacy, biology, electronics, power engineering, ecology, and many other fields. However, the complex engineering challenge here is the integration of functional nanostructures, such as molecular components (e.g., functional self-assembled molecular monolayers or entities of desoxyribonucleic acid, DNA), organic or inorganic nanoparticles (metal, semiconductor, colloidal materials), or similar building blocks into nanolithographically fabricated environments.Manufacturing paradigms for this integration go beyond current CMOS- and MEMS-fabrication schemes. Scale-bridging and hybrid manufacturing schemes that combine established top-down manufacturing techniques for microelectronic devices, including aspects of nanotechnology, with bottom-up nanoassembly of molecules on unstructured and pre-structured substrates are required. Microsystems technology based on the combination of conventional semiconductor processing with biotechnological approaches has thus become an emerging field with manifold applications, especially in the field of sensor technology.This contribution describes two illustrative and innovative research questions in nanopatterning and system integration at the micro-nano-interface to motivate applications with new and unprecedented functionalities. The first research question focuses on network-based biocomputing (NBC) using biological agents driven by biomolecular motor proteins in a lithographically fabricated nanofluidic channel system. This approach opens up opportunities for energy-efficient solutions to complex mathematical problems as they make their way through the NBC network. Methods are required to create switchable and rewritable nano-networks to address different mathematical problems using a single NBC-chip. Therefore, stimuli-responsive materials must be selectively and locally deposited into the nano-network to control the transport of the filaments through the network.The second research question focuses on nanophotonic devices based on DNA origami. The high-precision arrangement of nanoscopic substructures using specific binding sites (capture ends) of the DNA origami enables the precise arrangement of DNA origami functionalized with individualized metal nanoparticles. This arrangement could be used to exploit individual spectral response mediated by plasmonic effects.From a technological perspective, top-down lithographic nanostructuring technologies and bottom-up nanostructuring technologies are the basis for new device concepts. The adaptability of nanostructures to the world of biological molecules and other nanoscopic building blocks such as quantum dots and nanoparticles enables novel, even personalized, devices and engineering solutions.

Conformational Changes and ATP Hydrolysis in Zika Helicase: The Molecular Basis of a Biomolecular Motor Unveiled by Multiscale Simulations.

We computationally study the Zika NS3 helicase, a biological motor, using ATP hydrolysis energy for nucleic acid remodeling. Through molecular mechanics and hybrid quantum mechanics/molecular mechanics simulations, we explore the conformational landscape of motif V, a conserved loop connecting the active sites for ATP hydrolysis and nucleic acid binding. ATP hydrolysis, initiated by a meta-phosphate group formation, involves the nucleophilic attack of a water molecule activated by Glu286 proton abstraction. Motif V hydrogen bonds to this water via the Gly415 backbone NH group, assisting hydrolysis. Posthydrolysis, free energy is released when the inorganic phosphate moves away from the coordination shell of the magnesium ion, inducing a significant shift in the conformational landscape of motif V to establish a hydrogen bond between the Gly415 NH group and Glu285. According to our simulations, the Zika NS3 helicase acts as a ratchet biological motor with motif V transitions steered by Gly415's γ-phosphate sensing in the ATPase site.

Magnetic isotope effects and nuclear spin catalysis in living cells and biomolecular motors: recent advances and future outlooks: Nuclear spin catalysis.

Biomolecular nanoreactors are constructed from chemical elements many of which have magnetic and nonmagnetic stable isotopes. The magnetic isotope effects (MIE) were discovered in experiments with the cells enriched with different isotopes of magnesium, magnetic or nonmagnetic ones. The striking catalytic effect of the magnetic isotope, 25Mg, was revealed in the reaction of ATP hydrolysis driven by myosin, the biomolecular motor utilizing the chemical energy of ATP to perform the mechanical work. The rate of the enzymatic ATP hydrolysis with 25Mg as the enzyme cofactor is twice higher than the rates of the reactions with nonmagnetic 24Mg or 26Mg. A similar effect of the nuclear spin catalysis was revealed in the experiments with zinc as the myosin cofactor. MIE unambiguously indicate that, in the chemo-mechanical process catalyzed by the molecular motor, there is a limiting step which depends on the electron spin state of the reagents, and this step is accelerated by the nuclear spin of the magnetic isotope. The recent developments in this field highlight promising venues for future research of MIE in biophysics with possible applications of the magnetic isotopes in medical physics including radiation medicine and biomedical effects of electromagnetic fields.

In Vitro Synthesis and Design of Kinesin Biomolecular Motors by Cell-Free Protein Synthesis.

Kinesin is a biomolecular motor that generates force and motility along microtubule cytoskeletons in cells. Owing to their ability to manipulate cellular nanoscale components, microtubule/kinesin systems show great promise as actuators of nanodevices. However, classical in vivo protein production has some limitations for the design and production of kinesins. Designing and producing kinesins is laborious, and conventional protein production requires specific facilities to create and contain recombinant organisms. Here, we demonstrated the in vitro synthesis and editing of functional kinesins using a wheat germ cell-free protein synthesis system. The synthesized kinesins propelled microtubules on a kinesin-coated substrate and showed a higher binding affinity with microtubules than E. coli-produced kinesins. We also successfully incorporated affinity tags into the kinesins by extending the original sequence of the DNA template by PCR. Our method will accelerate the study of biomolecular motor systems and encourage their wider use in various nanotechnology applications.

Synthesis and characterization of (Co1-x Nix)3(BTC)2.12H2O (0 ≤ x ≤ 0.5) MOF based Janus chemical micromotors

We report the synthesis and characterization of Metal–Organic Framework (MOF)-Based Janus chemical Micro motors that self-propel at 26 and 25 µms−1 in 5% and 12% of H2O2, respectively, via ionic diffusiophoresis. The synthesis of this MOF was achieved by solvothermal method. The TG -DT analysis study on (Co1-x Nix)3(BTC)2·12H2O (0 ≤ x ≤ 0.5) micro motors, showed that water ligands can be easily removed. The Co3(BTC)2·12H2O crystallized in orthorhombic crystal system with Pmmm space group and (Co0.8Ni0.2)3(BTC)2·12H2O, (Co1.5Ni1.5)3(BTC)2·12H2O crystallized in monoclinic crystal system with C2 space group. The lattice constants, dislocation density and micro strain of the micro motors were calculated and the structure is discussed in detail using crystal viewer. The morphology of (Co1-x Nix)3(BTC)2·12H2O (0 ≤ x ≤ 0.5) micro motors were scrutinized using the field emission scanning electron microscopy (FE-SEM). Energy dispersion X-ray (EDX) analysis was employed for the elemental analysis and chemical characterization. The vibrational characterization was studied by utilizing Fourier Transform Infrared Spectroscopy (FT-IR) and Raman spectroscopy. The asymmetric and symmetric stretching vibrations of 1,3,5 trisubstituted aromatic ring appeared as three bands at 1368, 1428 and 1219 cm−1. The optical properties were explored using UV–Visible DRS and the absorption edge of Co-BTC, Co0.8Ni0.2-BTC and Co0.5Ni0.5-BTC were observed at approximately 300 nm, 450 nm and 500 nm respectively. The direct band gap for (Co1-x Nix)3(BTC)2.12 H2O (0 ≤ x ≤ 0.5) micromotors was found to be 3.84 eV, 3.76 eV and 3.66 eV respectively. The pore-size distribution and specific surface areas were calculated using Barrett–Joyner–Halenda, BJH and Brunauer Emmett-Teller method respectively. The average pore diameter 8.727 nm and total pore volume of 2.467 × 10−2cm3g−1 was obtained for the material. Insertion of nickel in the lattice of cobalt further accelerates the speed from 0.08 µms−1 to 26µms−1, which leads to a force of 7.29 fN and a power of 4.9 × 10−9 fW, which resembles bio molecular motors. Considering the investigation of MOFS as Microbots/Micromotors so far, Co0.8Ni0.2BTC in both 5% and 12% H2O2 can be represented as the best example of clear observation of bubble tails propelling spherical Janus micromotors.

Making motors work - potential applications in biocomputing and synthetic biology.

Biomolecular motors exhibit outstanding functions, including efficient motion and force generation, as well as autonomous operation. In this Essay, I discuss how biomolecular motors can be engineered to be used in artificial systems and what future applications such systems might have.

Rotary biomolecular motor-powered supramolecular colloidal motor.

Cells orchestrate the motion and force of hundreds of protein motors to perform various mechanical tasks over multiple length scales. However, engineering active biomimetic materials from protein motors that consume energy to propel continuous motion of micrometer-sized assembling systems remains challenging. Here, we report rotary biomolecular motor-powered supramolecular (RBMS) colloidal motors that are hierarchically assembled from a purified chromatophore membrane containing FOF1-ATP synthase molecular motors, and an assembled polyelectrolyte microcapsule. The micro-sized RBMS motor with asymmetric distribution of FOF1-ATPases can autonomously move under light illumination and is collectively powered by hundreds of rotary biomolecular motors. The propulsive mechanism is that a transmembrane proton gradient generated by a photochemical reaction drives FOF1-ATPases to rotate for ATP biosynthesis, which creates a local chemical field for self-diffusiophoretic force. Such an active supramolecular architecture endowed with motility and biosynthesis offers a promising platform for intelligent colloidal motors resembling the propulsive units in swimming bacteria.

3D Structure of Ring-shaped Microtubule Swarms Revealed by High-speed Atomic Force Microscopy

Biomolecular motor, microtubule (MT) and kinesin based molecular swarm robots are important due to their applications in cooperative task achievements, while the structural details are still unknown. In this work, high-speed atomic force microscopy was used to observe the MT swarm ring structure at nanometer-level resolution. MTs were observed to pile up in multiple layers to form swarm rings. The number of MTs involved in force generation was estimated which will specifically impact the understanding of the force-generation capability of swarms.

Directional transport of two-dimensional coupled Brownian particles subjected to nonconserved forces

<sec>In recent years, biomolecular motors have received widespread attention. Biomolecular motors are biological macromolecules that convert chemical energy into mechanical energy. The biomolecular motor is only a few tens of nanometers in size. According to Brownian theory of motion, people have constructed different types of Brownian ratchet models, such as rocking ratchets and closed-loop control ratchets. In previous studies, the directional transportation of Brownian ratchet is affected by conservative forces. These conservative forces include constant force, elastic force or harmonic force. However, whether the ratchet system can produce directional motion under the action of non-conservative forces is still rarely studied. Owing to the complex environment in the organism, for the studies of two-dimensional systems, the transport behavior of molecular motor has not been fully explained. Therefore, it is more practical to study the transport of Brownian particles in a two-dimensional ratchet potential.</sec><sec>The directional transport of two-dimensional Brownian particles subjected to conservative forces and non-conservative forces are studied in this work. It is found that the non-conservative external force has the effect of promoting the directional transport of coupling ratchets. With the change of the free length of the spring and spring coefficient, the average velocity of the coupled particles can be reversed. This means that the coupling effect can induce the inversion of two-dimensional Brownian ratchets. At the same time, the reverse transportation of coupled particles is enhanced under the interaction effect of conservative forces (spring elasticity) and non-conservative external forces. By choosing different kinds of external forces (conserved and non-conserved), in the experiment, it is possible to provide new method of separating two-dimensional coupled Brownian particles.</sec>

Rotations of F-ATPase and V-ATPase analyzed by a torque approach

F-type ATP synthase (F-ATPase) and vacuolar ATP hydrolase (V-ATPase) are well-known biomolecular motors, which play significant catalytic roles in ATP synthesis and ATP hydrolysis reactions. Their rotational torques are important factors involved in their rotational behavior that can be measured experimentally but with considerable difficulty. To overcome this difficulty and thereby provide an in-depth understanding of their operation mechanism, we herein carry out simple and fast computer modelling to study the two proteins, using our torque approach that relies on interatomic forces and coordinates of unequilibrated configurations taken from brief molecular dynamics (MD) simulations. As predicted by the torque approach, F-ATPase is demonstrated to be a random rotor, but it prefers to rotate in clockwise direction (as seen from the membrane toward the protein) for ATP synthesis, owing to the predominantly negative angle-averaged torques. By contrast, V-ATPase tends to rotate only in counterclockwise direction for ATP hydrolysis, due to the almost uniform averaged positive torques generated by the unidirectional rotation near the three catalytic sites. The rotational behaviors of both proteins are also affected by the surrounding solvent which can promote or hinder the internal rotation. By combining the torque approach with classic force-field MD simulations, the torques of two biomolecular motors can be calculated economically, and are found to agree with previous experiments and theoretical calculations. This work demonstrates that our torque approach can be extended to the field of biology and can help gain a deeper insight into the mechanistic rotation of biomolecular motors with modest computation time. Communicated by Ramaswamy H. Sarma

Solving the 3‐Satisfiability Problem Using Network‐Based Biocomputation

The 3‐satisfiability Problem (3‐SAT) is a demanding combinatorial problem that is of central importance among the nondeterministic polynomial (NP) complete problems, with applications in circuit design, artificial intelligence, and logistics. Even with optimized algorithms, the solution space that needs to be explored grows exponentially with the increasing size of 3‐SAT instances. Thus, large 3‐SAT instances require excessive amounts of energy to solve with serial electronic computers. Network‐based biocomputation (NBC) is a parallel computation approach with drastically reduced energy consumption. NBC uses biomolecular motors to propel cytoskeletal filaments through nanofabricated networks that encode mathematical problems. By stochastically exploring possible paths through the networks, the cytoskeletal filaments find possible solutions. However, to date, no NBC algorithm for 3‐SAT has been available. Herein, an algorithm that converts 3‐SAT into an NBC‐compatible network format is reported and four small 3‐SAT instances (with up to three variables and five clauses) using the actin–myosin biomolecular motor system are experimentally solved. Because practical polynomial conversions to 3‐SAT exist for many important NP complete problems, the result opens the door to enable NBC to solve small instances of a wide range of problems.

Converting microscale linear to rotary motion in kinesin-powered systems

Converting linear to rotary motion is a typical and enabling task for macroscale machinery, but the process is not yet established at the nano- and microscale. Linear motion is generated by the motor proteins kinesin and dynein as they move along microtubules, and a microtubule immobilized on the circumference of a microsphere could create rotary motion as it is propelled by surface-adhered motors, enabling microscale machines such as “active ball bearings”. This paper describes the attachment of microtubules to microspheres and the resulting types of motion as the microtubule-microsphere assemblies interact with a surface covered with kinesin-1 motor proteins. Dragging, pushing, spinning and rotating motions are observed and analyzed, demonstrating a pathway to microscale rotations driven by linear biomolecular motors.

Tuning the Force, Speed, and Efficiency of an Autonomous Chemically Fueled Information Ratchet.

Autonomous chemically fueled molecular machines that function through information ratchet mechanisms underpin the nonequilibrium processes that sustain life. These biomolecular motors have evolved to be well-suited to the tasks they perform. Synthetic systems that function through similar mechanisms have recently been developed, and their minimalist structures enable the influence of structural changes on machine performance to be assessed. Here, we probe the effect of changes in the fuel and barrier-forming species on the nonequilibrium operation of a carbodiimide-fueled rotaxane-based information ratchet. We examine the machine’s ability to catalyze the fuel-to-waste reaction and harness energy from it to drive directional displacement of the macrocycle. These characteristics are intrinsically linked to the speed, force, power, and efficiency of the ratchet output. We find that, just as for biomolecular motors and macroscopic machinery, optimization of one feature (such as speed) can compromise other features (such as the force that can be generated by the ratchet). Balancing speed, power, efficiency, and directionality will likely prove important when developing artificial molecular motors for particular applications.

In situ integrated microrobots driven by artificial muscles built from biomolecular motors.

Microrobots have been developed for applications in the submillimeter domain such as the manipulation of micro-objects and microsurgery. Rapid progress has been achieved in developing miniaturized components for microrobotic systems, resulting in a variety of functional microactuators and soft components for creating untethered microrobots. Nevertheless, the integration of microcomponents, especially the assembly of actuators and mechanical components, is still time-consuming and has inherent restrictions, thus limiting efficient fabrications of microrobots and their potential applications. Here, we propose a method for fabricating microrobots in situ inspired by the construction of microsystems in living organisms. In a microfluidic chip, hydrogel mechanical components and artificial muscle actuators are successively photopatterned from hydrogel prepolymer and biomolecular motors, respectively, and integrated in situ into functional microrobots. The proposed method allows the fast fabrication of microrobots through simple operations and affordable materials while providing versatile functions through the precise spatiotemporal control of in situ integration and reconfiguration of artificial muscles. To validate the method, we fabricated microrobots to elicit different motions and on-chip robots with unique characteristics for microfluidic applications. This study may establish a new paradigm for microrobot integration and lead to the production of unique biohybrid microrobots with various advantages.