Article pubs.acs.org/accounts
Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX
Molecular Assembly of Rotary and Linear Motor Proteins Yi Jia† and Junbai Li*,†,‡ †
Acc. Chem. Res. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 03/18/19. For personal use only.
Beijing National Laboratory for Molecular Sciences, CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China CONSPECTUS: Molecular machines are an important and emerging frontier in research encompassing interdisciplinary subjects of chemistry, physics, biology, and nanotechnology. Although there has been major interest in creating synthetic molecular machines, research on natural molecular machines is also crucial. Biomolecular motors are natural molecular machines existing in nearly every living systems. They play a vital role in almost every essential process ranging from intracellular transport to cell division, muscle contraction and the biosynthesis of ATP that fuels life processes. The construction of biomolecular motor-based biomimetic systems can help not only to deeply understand the mechanisms of motor proteins in the biological process but also to push forward the development of bionics and biomolecular motorbased devices or nanomachines. From combination of natural biomolecular motors with supramolecular chemistry, great opportunities could emerge toward the development of intelligent molecular machines and biodevices. In this Account, we describe our efforts to design and reconstitute biomolecular motor-based active biomimetic systems, in particular, the combination of motor proteins with layer-by-layer (LbL) assembled cellular structures. They are divided into two parts: (i) reconstitution of rotary molecular motor FOF1-ATPase, which is coated on the surface of LbL assembled microcapsules or multilayers and synthesizes adenosine triphosphate (ATP) through creating a proton gradient; (ii) molecular assembly of linear molecular motors, the kinesin-based active biomimetic systems, which are coated on a planar surface or LbL assembled tubular structure and drive the movement of microtubules. LbL assembled structures offer motor proteins with an environment that resembles the natural cell. This enables high activity and optimized function of the motor proteins. The assembled biomolecular motors can mimic their functionalities from the natural system. In addition, LbL assembly provides facile integration of functional components into motor protein-based active biomimetic systems and achieves the manipulation of FOF1-ATPase and kinesin. For FOF1-ATPase, the light-driven proton gradient and controlled ATP synthesis are highlighted. For kinesin, the strategies used for the direction and velocity control of kinesin-based molecular shuttles are discussed. We hope this research can inspire new ideas and propel the actual applications of biomolecular motor-based devices in the future.
1. INTRODUCTION Movement is essential to life. All life activities, for example, muscle contraction, intracellular transport, genetic material (deoxyribonucleic acid, DNA) replication and cell division, are derived from the propulsion of motor proteins at the molecular level. Motor proteins or molecular motors are several classes of biological molecules that distribute in the interior or on the surface of a cell, which can transform chemical energy into mechanical work. Molecular motors can be divided into two categories: linear molecular motors and rotary molecular motors.1 Linear molecular motors mainly consist of kinesin, myosin, and dynein. These linear molecular motors can effectively and directly convert chemical energy obtained by the hydrolysis of adenosine triphosphate (ATP) into mechanical energy and move on their associated filaments directionally. For example, myosin walks along actin filaments, while kinesin and dynein move along microtubules. Rotary molecular motors comprise ATPase and bacterial flagella. They are powered by an ion gradient, using both electric and entropic forces. These two kinds of molecular motors both © XXXX American Chemical Society
exhibit high energy-conversion efficiencies compared to manmade devices, which, together with their nanoscale, prompted a large number of studies over the past two decades.1−6 These studies are aimed at uncovering the fundamental mechanisms of molecular motors and the integration of molecular motors in hybrid micro- and nanodevices. Supramolecular chemistry provides diverse strategies to design and construct highly advanced functional materials systems.7 From combination of natural biomolecular motors with supramolecular chemistry, great opportunities could emerge toward the development of intelligent molecular machines. In this Account, we describe our efforts on the in vitro reconstitution and integration of molecular motors (FOF1ATPase and kinesin) in artificial biomimetic systems. In particular, the combination of molecular motors with layer-bylayer (LbL) assembled cellular structures is highlighted, and the coupling with functional components for the feasible Received: January 8, 2019
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DOI: 10.1021/acs.accounts.9b00015 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 1. (A) Schematic illustration, CLSM image, and AFM image of FOF1-ATPase in hollow lipid bilayer-coated polyelectrolyte microcapsule. Reproduced with permission from ref 11. Copyright 2007 Wiley-VCH. (B) Schematic illustration of natural plant cell, chloroplast, and the main protein components in thylakoid stacking structure. (C) Schematic illustration and SEM images of honeycomb CA film, CA/(PEI/PSII)n/PEI, CA/(PEI/PSII)5/PEI/ATPase−liposome. Reproduced with permission from ref 14. Copyright 2018 American Chemical Society. (D) SEM image, schematic illustration, and TEM image of PSII-sol−gel@polyelectrolyte microtube array. Reproduced with permission from ref 15. Copyright 2018 Wiley-VCH.
ATP from ADP and inorganic phosphate (Pi) by using a transmembrane proton gradient. The reconstitution of FOF1ATPase in artificial assembled systems not only can well exploit its rotational mechanical energy but also can mimic a cellular process to synthesize “energy currency”, ATP, in vitro. More importantly, it provides the opportunity to break the limit of natural reactions and achieves output control.
manipulation of these molecular motors is discussed. We hope this Account can help to better understand the molecular motor-based biomimetic systems, inspire new ideas, and promote the development of biomolecular motor-based devices or nanomachines.
2. FOF1-ATPase-BASED ACTIVE BIOMIMETIC ASSEMBLY FOF1-ATPase is an energy-linked enzyme that widely exists in the cell membranes of living organism.8 It plays vital roles in cellular energy interconversion that either pumps protons across a membrane by using ATP hydrolysis or synthesizes
2.1. Reconstitution of FOF1-ATPase in LbL Assembled Cellular Structures
FOF1-ATPase is a membrane-bound protein. Embedding of FOF1-ATPase in the membrane structures and a transB
DOI: 10.1021/acs.accounts.9b00015 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 2. (A) Schematic illustration and (B) CLSM image of FOF1-ATPase proteoliposomes on PSII-based microspheres. The fluorescence signal is attributed to the chlorophylls in PSII (green) and Texas-red labeled lipid (red). (C) Schematic illustration of light-induced water splitting and proton production by PSII. (D) Light-induced proton gradient and (E) ATP synthesis within FOF1-ATPase proteoliposomes coated PSII-based microspheres under light illumination (red line) and in the dark (blue line). (F) ATP synthesis through on−off cycles of light. Reproduced with permission from ref 27. Copyright 2015 American Chemical Society. (G) Schematic illustration of FOF1-ATPase proteoliposomes on 1-hydroxy pyrene (PyOH) entrapped multilayers. (H) Light-driven pH changes and (I) ATP synthesis. Reproduced with permission from ref 16. Copyright 2017 American Chemical Society.
coated polyelectrolyte microcapsules.11 The oppositely charged poly(acrylic acid) (sodium salt) (PAA) and poly(allylamine hydrochloride) (PAH) were chosen as the wall components and alternately adsorbed onto the surfaces of sacrificial template particles via electrostatic interactions. Removal of the template yield hollow microcapsules. The microcapsules have similar morphology to natural cells (Figure 1A) and act as a robust support for FOF1-ATPase-containing liposomes. Moreover, these hollow microcapsules not only can serve as reactors for the synthesis of ATP but also can act as bioenergy containers for the storage of ATP. Subsequent works replace polyelectrolytes with natural proteins to improve the properties of microcapsules.12,13 For example, glucose oxidase (GOx) microcapsules were prepared through covalent LbL assembly by using glutaraldehyde (GA) as a cross-linker.12 In comparison with polyelectrolyte microcapsules, GOx microcapsules have better biocompatibility and better stability due to the covalent assembly. In addition, the transmembrane proton gradient can be directly generated by GOx catalyzed oxidation of glucose in the bulk solution. To better mimic the real cellular environment, we recently constructed an artificial thylakoid through the combination of breath figure method and LbL assembly.14 The breath figure method is frequently used to prepare highly ordered
membrane proton gradient are prerequisites for FOF1-ATPase to fulfill its function of ATP synthesis. In the living organism, the cell membrane is formed by the lipid self-assembly. Therefore, lipid membrane was well recognized as a model for biomembranes and was chosen as useful platforms for in vitro protein reconstitution in basic structural and functional studies of membrane proteins. Earlier studies were mostly interested in the incorporation of FOF1-ATPase in the surface of liposomes, which have the most structural similarity to the natural cell membranes.9 The use of polymersomes in fact improved the stability of FOF1-ATPase-based systems.10 With the development of layer-by-layer constructed microcapsules, our group has started to integrate FOF1-ATPase-incorporated proteoliposomes in a microstructured surface, which is closer to the real cell size and functions for the manipulation of ATP synthesis.11−16 Such assembled micro- or nanostructures serve as robust supports for proteoliposomes, which can greatly improve the stability of the system. Moreover, they can be constructed with a variety of different components and driving forces including the covalent binding.17,18 It can also endow the assembled systems with tunable physicochemical properties such as size, morphology, stability, and permeability.18−20 This has been demonstrated by our first report on the reconstitution of FOF1-ATPase onto the lipid bilayerC
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Accounts of Chemical Research honeycomb-patterned films. The principle of this method is solvent evaporation induced water condensation and the subsequent use of condensed water droplets as a dynamic template. To prepare cellulose acetate (CA) honeycomb membrane, CA was dissolved in a mixture of acetone and dichloromethane and then cast on the glass substrate. The evaporation of dichloromethane decreased the solution surface temperature, which caused water vapor to condense and selforganize into a hexagonal pattern. After complete evaporation of the organic solvents and the water droplets, honeycomb-like CA membrane was obtained. The honeycomb-like CA membrane is analogous to natural thylakoid grana structure (Figure 1B,C) and provides enough space for the assembly of functional components. Polyethylenimine (PEI) and photosystem II (PSII) were alternately deposited into the cavities of the CA honeycomb membrane (Figure 1C). The obtained PEI/PSII multilayers were shown to well imitate the stacking structure of the natural thylakoid (Figure 1B). More importantly, the ATP production can be easily manipulated by changing the numbers of assembled bilayers. Another work reported a hierarchical ordered and compartmentalized structures resembling to palisade-like tissue in leaves (Figure 1D).15 In this system, cable-like polyelectrolyte multilayer microtube was used as a scaffold, mimicking the shell of a cylindrical cell in natural plant leaves. PSII and FOF1-ATPase are compartmentally localized in the inner space and the surface of polyelectrolyte multilayer microtube, respectively. Such hierarchical compartmentalized structures are shown to facilitate the phosphorylation and contribute to high phosphorylation efficiency, which is about 14 times higher than natural chloroplast. These compartmentalization and integration strategies may open a new approach for coassembly of multicomponents to mimic vital biochemical processes and achieve remote control.
membrane and generates proton gradients to drive ATP synthesis. For higher plants, photophosphorylation is a complicated procedure accomplished by the coordination of photosystem II (PSII), photosystem I (PSI), cytochrome b6f complex, and FOF1-ATPase. PSII catalyzes the initial step of photosynthesis that drives oxidation of water into protons, electrons, and oxygen.24 Therefore, PSII is an excellent building block that can utilize solar energy to split water into proton and electron sources.25,26 Considering this, we took the first attempt to integrate PSII with FOF1-ATPase in a welldefined core@shell structure (Figure 2A,B).27 PSII-based microspheres serving as core were prepared via coprecipitation method and subsequently cross-linked with GA. The shell that consists of FOF1-ATPase proteoliposomes was then coated on the surface of PSII microspheres to establish a simple chloroplast-like system. The PSII trapped in the microspheres was shown to retain its activity and produced protons under red light irradiation. In this way, a transmembrane proton gradient was created between the interior and the exterior of PSII microspheres (Figure 2C,D). The light-induced proton gradient subsequently drives the rotational catalysis of FOF1ATPase to produce ATP. The concentration of ATP was shown to increase with the increase of irradiation time, while it was nearly unchanged in the dark (Figure 2E). This artificial chloroplast-like assembled system makes it possible to decouple the natural photophosphorylation pathway and achieves light-driven ATP synthesis bypassing cytochrome b6f and PSI. More importantly, ATP synthesis can be facilely manipulated by switching light on and off (Figure 2F). This is critical for smart nanodevices in practical application. However, it should be mentioned that the ATP production is not high enough for powering certain systems or devices. Therefore, we recently devoted efforts to the enhancement of the photophosphorylation efficiency in assembled biomimetic systems. One strategy is to construct the structures that much closer to the plant cells, such as thylakoid-like and palisade-like structures.14,15 PSII and FOF1-ATPase are spatially arranged in these hierarchical structures. These structures provide the compartmentalized space for the enzyme reaction to take place and enable locally high proton gradient. This facilitates photophosphorylation and thus produces ATP with high efficiency. Another strategy involves the direct modification of chloroplasts. In natural chloroplast, the light harvested by PSII is limited to visible regions. To extend the range of solar light utilization, artificial chloroplasts or chloroplast hybrid systems have been developed.28−30 Colloidal semiconductor quantum dots (QDs) possess excellent optical properties, and their photoluminescence can be easily tuned by precisely controlling their size, composition, and shape. We synthesized optically matched CuInS 2 /ZnS QDs and entrapped them into chloroplasts to increase the light absorption range of PSII.29 Under light illumination, CuInS2/ZnS QDs strongly absorb incident light, especially ultraviolet (UV) light and then emit red light that can be efficiently captured by PSII. In this way, PSII captured more light to oxidize water, which produced more protons as compared to that of natural chloroplasts, and therefore distinctly improved the photophosphorylation efficiency. Aside from natural protein PSII, using an appropriate proton generator is a more convenient method for producing proton. Photoacid generators are a class of proton-bearing small molecules that release protons in the photoexcited state and create a rapid pH jump.31 We chose 1-hydroxy pyrene
2.2. ATP Synthesis from FOF1-ATPase-Based Active Biomimetic Assembly
The reconstitution of FOF1-ATPase in the membrane structures offer the premise for ATP synthesis, while a transmembrane proton gradient is another essential prerequisite. Previous studies mostly adopt acid−base transition to create a proton gradient.11 However, the produced proton gradient can only be maintained for a few minutes. In our earlier studies, we tried to create a transmembrane proton gradient by the oxidation of glucose.12,13 This proton gradient can remain for a few hours. Recently, there has been tremendous interest in constructing smart molecular systems that are capable of performing specific tasks in response to a certain signal input.21 As a control signal, light is particularly attractive because it can be delivered with high spatiotemporal precision, which enables excellent controllability. Herein, we highlight the strategies used for light-induced proton gradient and our efforts on the enhancement of photophosphorylation efficiency. Photosynthesis is a process in green plants and other organisms that converts solar energy into chemical energy, which can be later used to fuel diverse activities of organisms. Photophosphorylation is a vital part of photosynthesis. It utilizes light as a source of energy and ultimately converts ADP to ATP. Previous studies focused on the coupling of bacteriorhodopsin (BR) and FOF1-ATPase in liposomes to mimic the photophosphorylation in the Archaea.22,23 Upon light illumination, BR translocates protons across the lipid D
DOI: 10.1021/acs.accounts.9b00015 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 3. (A) Schematic illustration of a capsule filled with FITC-dextran transported by kinesin along a microtubule. Reproduced with permission from ref 38. Copyright 2008 Elsevier. (B) Schematic illustration and (C−F) time-lapse images of a capsule transported by a microtubule moved on the kinesin-modified surface. The scale bar represents 5 μm. Reproduced with permission from ref 39. Copyright 2009 Elsevier.
steadily, while no ATP was produced without MGCB nanoparticles. This proved the vital role of MGCB in the ATP synthesis and the feasibility of using photobase generators to create proton gradients. The strategies introduced above achieve light-induced proton gradient and propel the consequent ATP synthesis. The light-driven ATP production in spatially defined regions provides a facile means of harnessing ATP-consumed devices or nanomachines, such as motor protein kinesin-based nanoscale transport systems. This is another important direction that utilizing motor proteins to transform the chemical energy of ATP into mechanical energy.
(PyOH) molecules as photoacid generators and entrapped them in the LbL assembled PAH/graphene oxide (GO) multilayers (Figure 2G).16 Under light illumination, PyOH in the PAH/GO film rapidly dissociated into PyO− and H+, resulting in a distinct pH decrease in the system. The ΔpH value was shown to depend on the quantity of PyOH, which increases with the number of assembled layers (Figure 2H). For (PAH/GO)6−PyOH, the pH declines quickly from 6.3 to 3.8 within 5 min. The rapid formation of a transmembrane proton gradient leads to remarkable enhancement of the photophosphorylation efficiency (Figure 2I). Based on this work, we further attempt to directly introduce photoacid generator into thylakoid membranes of natural chloroplasts.30 To increase the lifetime of the proton dissociation state and enable a good match with chloroplast, long-lived photoacid protonated merocyanine (MEH) was encapsulated into the thylakoid lumen by ultrasonication. Compared to natural chloroplasts, the MEH−chloroplast hybrid system produces more protons by both PSII and MEH, thus dramatically augmenting the photophosphorylation efficiency for ATP synthesis, which is up to 3.9 times beyond natural chloroplasts. In contrast to photoacid generators, photobase generators provide an alternative way to create a proton gradient based on the principle of proton-consumption. Under light illumination, photobase generator molecules yield OH− that can be used to neutralize local H+, thus producing a proton gradient. As a proof of demonstration, malachite green carbinol base (MGCB) was used as base source.32 MGCB was assembled into nanoparticles through phase transfer to improve its solubility. When the MGCB nanoparticles were exposed to UV light, they release OH− and malachite green cation (MG+). An increase of the pH value and an obvious color change were observed, demonstrating the excellent pH regulation ability of MGCB nanoparticles. The MGCB nanoparticles were then incubated with acid buffer containing FOF1-ATPase-liposomes. Under UV light irradiation, ATP concentration was increased
3. KINESIN-BASED ACTIVE BIOMIMETIC ASSEMBLY Kinesins are eukaryotic microtubule-associated motor proteins that primarily involved in intracellular transport, cell division, and the organization of cilia and flagella.33 They can transform the chemical energy of ATP into mechanical work, moving linearly along a microtubule. The energy conversion efficiency of kinesin is up to 50%, which is twice as efficient as the heat engine used in cars.34 This makes the kinesin molecule an attractive assembly unit in artificial molecular devices. With the development of molecular biology and modern biological separation technology, researchers directly purified kinesin and microtubule and reconstituted them in vitro to mimic their biological functions, in particular their function as nanoscale transport system (termed “molecular shuttles”). 3.1. Construction of Kinesin-Based Molecular Shuttles
Kinesin-based molecular shuttles comprise two geometries: “bead geometry” and “gliding geometry”. In the bead geometry, microtubules are immobilized by adsorption or chemical bonding to a surface, and kinesins slide along the microtubule. In the gliding geometry (or “inverted geometry”), kinesins are immobilized and microtubules glide on kinesin functionalized surfaces. For cargo transport, previous studies E
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Figure 4. (A) Illustration of LbL-assembled tubes as tracks to guide the movement of microtubules. (B−G) Time-lapse images of microtubule movement in the inner wall of the nonfunctionalized tube (B−D) and Ni−NTA functionalized tube (E−G). The white arrows point to the leading head of the microtubule. Reproduced with permission from ref 47. Copyright 2014 American Chemical Society.
mostly adopt polystyrene beads,35 quantum dots,36 and magnetic nanoparticles37 as cargo models. In order to expand the diversity of cargos and achieve controlled cargo loading and unloading, we combined LbL assembled microcapsules or microspheres with kinesin-based molecular shuttles and employed these structures as carriers for cargo transport.38−41 The versatile LbL assembly technique can allow a large variety of cargos to be assembled or entrapped into nanoscale films of microcapsules or microspheres, such as polyelectrolytes, proteins, polysaccharides, inorganic particles, dendrimers, vesicles, micelles, virus particles, and so on.17−20 More importantly, the diverse wall components and different driving forces enable the LbL assembled capsules to have various stimuli-responsivity, which allows controlled release (unloading) of cargos from the capsules upon specific stimuli.17,18 As a model cargo, fluorescein isothiocyanate-dextran (FITC-dextran) was loaded into LbL assembled polyelectrolyte microcapsules via co-incubation. The obtained capsules can be easily functionalized by kinesin via electrostatic interaction or be attached to the microtubules via biotin−streptavidin linkage. Thus, “bead geometry” (Figure 3A) and “gliding geometry” (Figure 3B) based molecular shuttles were constructed, respectively.38,39 Taking the “gliding geometry” as an example, the microcapsules were first modified with streptavidin, and then bound to the biotinylated microtubules.39 The microcapsule−microtubule complex was added into a kinesin-coated chamber. In the presence of ATP, both microtubules and microcapsule−microtubule complex moved on the kinesincoated surface (Figure 3C−F). Further detailed analysis revealed that the capsule sizes did not affect the gliding velocity of microtubules, but the probability of capsule motility correlated with the size of capsule and the proportion of biotinylation on the microtubule. Despite kinesin-based molecular shuttles being reconstructed in vitro and some systems already having been
integrated in primitive nanodevices for diverse applications,3 two main technological problems still need to be resolved for the successful realization of future smart nanodevices: (i) achievement of unidirectional movement (direction control) of kinesin or microtubule; (ii) on−off control (velocity control) of the kinesin−microtubule system. 3.2. Direction Control of Kinesin-Based Molecular Shuttles
The random motion of microtubules has always been a major obstacle in the practical application of kinesin-powered nanodevices. Surface chemistry is a simple method that directly constructs patterns of kinesins on the substrates, which force the microtubules to move along the patterns in the desired direction. Recently, we constructed hydrophobic/ hydrophilic partitions on a glass substrate through tertbutoxycarbonyl (Boc) protection and selective deprotection of amines.42 An aminated glass substrate was first exposed to di-tert-butyl dicarbonate ((Boc)2O) solution. The formation of N-Boc groups endowed the glass substrate with high hydrophobicity. Then, selective cleavage of the N-Boc group (deprotection) using hydrochloric acid was conducted to produce amino groups on the specified region. In this way, hydrophobic/hydrophilic partitions were constructed on the glass substrate. Kinesins can be selectively immobilized on the hydrophilic regions. Thus, the microtubules were restricted to move in confined regions that were modified with kinesins. Other groups also developed the micro- or nanopatterns using UV lithography or electron beam lithography for kinesin-based molecular shuttles.3 However, it was found that when the filaments reached the boundary of the patterns, they may easily detach from the surface. Therefore, surface chemistry is often used together with other approaches, such as surface topography and external forces.43,44 In recent years, the concept of employing linear tubes as tracks was proposed to direct the movement of filaments or F
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Figure 5. (A) Schematic illustration and (B−E) Time-lapse images of the self-powered CPK−microtubule complex gliding on the kinesin-modified surface. The scale bars represent 5 μm. Reproduced with permission from ref 40. Copyright 2015 Royal Society of Chemistry.
microtubules. Teizer and co-workers first demonstrated the using of the external surface of multiwalled carbon nanotube as a track and successfully guided the movement of microtubules.45 Considering that microtubules may detach or derail from the external surface of the tube, Lard and co-workers exploited the inner space of hollow Al2O3 nanowire as a track and realized unidirectional, one-dimensional motion of filaments inside the hollow nanowire.46 Recently, our group reported the exploration of LbL assembled tubes as a track for microtubule guiding (Figure 4).47 LbL assembled multilayer tubes were prepared by alternate deposition of oppositely charged polyelectrolytes into porous polycarbonate (PC) membrane and removal of the PC membrane. Then kinesins were adsorbed into the interior surface of the LbL assembled tube by co-incubation. As shown in Figure 4B−D, the kinesinadsorbed tube could confine the microtubules to move unidirectionally through the tube; however, the microtubules did not adhere well to the tube wall and slowly floated out of the tubes. This is because physically adsorbed kinesin may not be densely packed and is randomly distributed with either head or tail domains exposed. To obtain higher kinesin density with active microtubule binding sites exposed, Ni−NTA (nitrilotriacetic acid) complex was selectively attached onto the inner wall of a multilayer tube. The Ni−NTA complex can specifically bind to the histidine-tagged C-terminal end of kinesin, which leaves the N-terminal end free for microtubule binding.48 It is shown in Figure 4E−G that the microtubule tightly grasped the Ni−NTA functionalized tube wall and smoothly glided through the tubular channel. Taking advantage of the LbL assembly technique, both the channel size and the function of the assembled tube could be readily adjusted; for example, one can selectively modify inner walls or outer walls or both walls of the tube with active kinesins that both the interior and exterior of LbL assembled tubes can be used as tracks for microtubule guiding.
microtubules, respectively. However, it often requires additional microfluidic equipment and the exchange of solution may also result in replacement or loss of kinesins or microtubules;49 this is not suitable for practical applications. The ideal control is to switch ON/OFF the motility of kinesin or microtubules at any desired position and desired time. Stimuli-responsive ATP supply makes it possible and enables an elegant velocity control of kinesin-based molecular shuttles. Light is a frequently used signal for switching because it allows regulation with high temporal and spatial precision. Higuchi and co-workers initially demonstrated the light-controlled switching of kinesin’s motility from OFF to ON state by using a caged ATP.50 ATP was released by flash photolysis under UV light and induced the movement of kinesin. Later, lightcontrolled molecular shuttles were built.35 The motility of microtubule can be turned ON through UV-induced release of caged ATP and turned OFF through hexokinase degradation of ATP. Some studies also reported the using of electric fields for the release of trapped ATP or regulating the activity of kinesin to control the gliding speeds.51,52 Though the supply of ATP can be manipulated, repeated exposure to UV light and electrical fields may cause damage to the activity of motor proteins. By contrast, enzymatically induced ATP supply under mild conditions is more suitable for biomotor systems.40,49 Recently, we reported an ATP generation system based on creatine phosphate kinase (CPK) microspheres (Figure 5).40 The CPK microspheres were prepared by a coprecipitation method and subsequently cross-linked by GA. These CPK microspheres well retained their bioactivity that can catalyze the conversion of ADP and creatine phosphate to ATP. When the CPK microspheres were added, microtubules began to move on the kinesin-coated surface, and the velocity of microtubules increased with the increased concentration of produced ATP. More importantly, the increased ADP released during the motion of kinesins could be reused for ATP production. This could not only buffer the concentration of ATP in the kinesin−microtubule system but also avoid the inhibiting effects on the activity of kinesins induced by accumulated excess ADP. The coprecipitation method also enables the easy encapsulation of diverse cargos (such as GOx, FITC-dextran) into the CPK microspheres. As a demonstration, GOx-loaded CPK microspheres were fabricated and bound to the biotinylated microtubule via streptavidin−biotin interaction. With self-supplying ATP,
3.3. Velocity Control of Kinesin-Based Molecular Shuttles
To apply motor proteins as natural molecular machines for nanotransportation, complete temporal control over ON/OFF switching of the motility is necessary. For kinesin-based molecular shuttles, ATP is required as an energy source. Therefore, manipulating the supply of ATP is a direct route for velocity control of kinesin-based molecular shuttles. Obviously, the simplest method is directly adding ATP to and eliminating ATP from the system to switch ON and OFF the movement of G
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microtubule can drag the GOx-loaded CPK microspheres and move on the kinesin-modified surface (Figure 5B−E). The CPK microspheres here not only supply ATP to the system but also serve as cargo carriers, thus achieving the facile loading and self-powered delivery of cargos simultaneously. In the subsequent study, we also realized pH-responsive ATP supply for the kinesin−microtubule system by using LbL assembled multilayer coated CaCO3 microspheres as ATP carriers.41 In the acid environment, CaCO3 microspheres were gradually decomposed and released the entrapped ATP, which then propelled the movement of microtubules. Additionally, these porous CaCO3 microspheres could also load cargos and finally achieve pH-responsive molecular shuttles.
The authors declare no competing financial interest. Biographies Yi Jia is an associate professor in Prof. Li’s group at the Institute of Chemistry, the Chinese Academy of Sciences. Her research interests include layer-by-layer assembled micro- and nanostructures, reconstitution of motor proteins, and their related biomedical applications. Junbai Li is a Professor at the Institute of Chemistry, the Chinese Academy of Sciences. His research interests involve molecular biomimetics based on molecular assembly, reconstitution of motor proteins, self-assembly of dipeptides, biointerfaces, bioinspired materials, and nanostructured design.
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4. CONCLUSIONS AND PROSPECTS LbL assembly technology provides a unique opportunity to make use of motor proteins for the development and engineering of molecular devices. First, LbL assembled biomimetic membrane can offer motor proteins with an environment much more similar to the natural cell by simply changing the components and templates. This ensures higher activity and optimizes function of motor proteins in artificial biomimetic systems. Second, LbL assembled biomimetic membranes enable the facile encapsulation of functional components to smartly manipulate the functions of motor proteins, such as encapsulation of PSII or photoacid generators to achieve light-driven rotational catalysis of FOF1-ATPase and the entrapping of CPK in the microsphere to supply kinesins with energy ATP. As a result, LbL assembled biomimetic membranes offer remarkable benefits and tremendous possibilities over natural cell membranes. They provide a good platform for regeneration and efficient manipulation of motor proteins in an artificial environment. In spite of notable progress, biomolecular motor-based systems are still at an early stage of development. In the future, critical issues should be addressed before they achieve revolutionary practical applications. For the FOF1-ATPase, the orientation is random and not always as desired, and therefore it may not be appropriate for the expected function. New reconstitution strategies or membrane systems will need to be developed in order to control the orientation of reconstituted FOF1-ATPase in assembled systems. Concerns should also be directed to the properties (composition, curvature, permeability, etc.) of assembled membranes and their influence on the function of FOF1-ATPase. In addition, how to harness the energy of generated ATP and further energy transformation need to be considered. For kinesin, the smart spatial and temporal regulation, as well as controlled cargo loading and unloading by kinesins or microtubules, are still the important directions of future research. With the development of assembly strategies and improved understanding of biomolecular motors, we believe that the ongoing engineering of biomolecular motors will make a breakthrough and finally find its practical applications in the near future.
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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (Project Nos. 21433010, 21872151, and 21320102004).
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AUTHOR INFORMATION
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[email protected]. ORCID
Yi Jia: 0000-0001-9812-667X Junbai Li: 0000-0001-9575-3125 H
DOI: 10.1021/acs.accounts.9b00015 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Article
Accounts of Chemical Research
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DOI: 10.1021/acs.accounts.9b00015 Acc. Chem. Res. XXXX, XXX, XXX−XXX
