Synthetic Systems Powered by Biological Molecular Motors.

Biological molecular motors (or biomolecular motors for short) are nature's solution to the efficient conversion of chemical energy to mechanical movement. In biological systems, these fascinating molecules are responsible for movement of molecules, organelles, cells, and whole animals. In engineered systems, these motors can potentially be used to power actuators and engines, shuttle cargo to sensors, and enable new computing paradigms. Here, we review the progress in the past decade in the integration of biomolecular motors into hybrid nanosystems. After briefly introducing the motor proteins kinesin and myosin and their associated cytoskeletal filaments, we review recent work aiming for the integration of these biomolecular motors into actuators, sensors, and computing devices. In some systems, the creation of mechanical work and the processing of information become intertwined at the molecular scale, creating a fascinating type of "active matter". We discuss efforts to optimize biomolecular motor performance, construct new motors combining artificial and biological components, and contrast biomolecular motors with current artificial molecular motors. A recurrent theme in the work of the past decade was the induction and utilization of collective behavior between motile systems powered by biomolecular motors, and we discuss these advances. The exertion of external control over the motile structures powered by biomolecular motors has remained a topic of many studies describing exciting progress. Finally, we review the current limitations and challenges for the construction of hybrid systems powered by biomolecular motors and try to ascertain if there are theoretical performance limits. Engineering with biomolecular motors has the potential to yield commercially viable devices, but it also sharpens our understanding of the design problems solved by evolution in nature. This increased understanding is valuable for synthetic biology and potentially also for medicine.

Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.

Eg5 is a kinesin motor protein that is responsible for bipolar spindle formation and plays a crucial role during mitosis. Loss of Eg5 function leads to the formation of monopolar spindles, followed by mitotic arrest, and subsequent cell death. Several cell-permeable small molecules have been reported to inhibit Eg5 and some have been evaluated as anticancer agents. We now describe the design, synthesis, and biological evaluation of photoswitchable variants with five different pharmacophores. Our lead compound Azo-EMD is a cell permeable azobenzene that inhibits Eg5 more potently in its light-induced cis form. This activity decreased the velocity of Eg5 in single-molecule assays, promoted formation of monopolar spindles, and led to mitotic arrest in a light dependent way.

Microtubule Detachment in Gliding Motility Assays Limits the Performance of Kinesin-Driven Molecular Shuttles.

The creation of complex active nanosystems integrating cytoskeletal filaments propelled by surface-adhered motor proteins often relies on the filaments' ability to glide over up to meter-long distances. While theoretical considerations support this ability, we show that microtubule detachment (either spontaneous or triggered by a microtubule crossing event) is a non-negligible phenomenon that has been overlooked until now. The average gliding distance before spontaneous detachment was measured to be 30 ± 10 mm for a functional kinesin-1 density of 500 µm-2 and 9 ± 4 mm for a functional kinesin-1 density of 100 µm-2 at 1 mM ATP. Even microtubules longer than 3 µm detached, suggesting that spontaneous detachment is not caused by the stochastic absence of motors or their stochastic release due to a limited run length.

Engineering with Biomolecular Motors.

Biomolecular motors, such as the motor protein kinesin, can be used as off-the-shelf components to power hybrid nanosystems. These hybrid systems combine elements from the biological and synthetic toolbox of the nanoengineer and can be used to explore the applications and design principles of active nanosystems. Efforts to advance nanoscale engineering benefit greatly from biological and biophysical research into the operating principles of motor proteins and their biological roles. In return, the process of creating in vitro systems outside of the context of biology can lead to an improved understanding of the physical constraints creating the fitness landscape explored by evolution. However, our main focus is a holistic understanding of the engineering principles applying to systems integrating molecular motors in general. To advance this goal, we and other researchers have designed biomolecular motor-powered nanodevices, which sense, compute, and actuate. In addition to demonstrating that biological solutions can be mimicked in vitro, these devices often demonstrate new paradigms without parallels in current technology. Long-term trends in technology toward the deployment of ever smaller and more numerous motors and computers give us confidence that our work will become increasingly relevant. Here, our discussion aims to step back and look at the big picture. From our perspective, energy efficiency is a key and underappreciated metric in the design of synthetic motors. On the basis of an analogy to ecological principles, we submit that practical molecular motors have to have energy conversion efficiencies of more than 10%, a threshold only exceeded by motor proteins. We also believe that motor and system lifetime is a critical metric and an important topic of investigation. Related questions are if future molecular motors, by necessity, will resemble biomolecular motors in their softness and fragility and have to conform to the "universal performance characteristics of motors", linking the maximum force and mass of any motor, identified by Marden and Allen. The utilization of molecular motors for computing devices emphasizes the interesting relationship among the conversion of energy, extraction of work, and production of information. Our recent work touches upon these topics and discusses molecular clocks as well as a Landauer limit for robotics. What is on the horizon? Just as photovoltaics took advantage of progress in semiconductor fabrication to become commercially viable over a century, one can envision that engineers working with biomolecular motors leverage progress in biotechnology and drug development to create the engines of the future. However, the future source of energy is going to be electricity rather than fossil or biological fuels, a fact that has to be accounted for in our future efforts. In summary, we are convinced that past, ongoing, and future efforts to engineer with biomolecular motors are providing exciting demonstrations and fundamental insights as well as opportunities to wander freely across the borders of engineering, biology, and chemistry.

The Photosystem II D1-K238E mutation enhances electrical current production using cyanobacterial thylakoid membranes in a bio-photoelectrochemical cell.

The conversion of solar energy (SEC) to storable chemical energy by photosynthesis has been performed by photosynthetic organisms, including oxygenic cyanobacteria for over 3 billion years. We have previously shown that crude thylakoid membranes from the cyanobacterium Synechocytis sp. PCC 6803 can reduce the electron transfer (ET) protein cytochrome c even in the presence of the PSII inhibitor DCMU. Mutation of lysine 238 of the Photosystem II D1 protein to glutamic acid increased the cytochrome reduction rates, indicating the possible position of this unknown ET pathway. In this contribution, we show that D1-K238E is rather unique, as other mutations to K238, or to other residues in the same vicinity, are not as successful in cytochrome c reduction. This observation indicates the sensitivity of ET reactions to minor changes. As the next step in obtaining useful SEC from biological material, we describe the use of crude Synechocystis membranes in a bio-photovoltaic cell containing an N-acetyl cysteine-modified gold electrode. We show the production of significant current for prolonged time durations, in the presence of DCMU. Surprisingly, the presence of cytochrome c was not found to be necessary for ET to the bio-voltaic cell.

Effect of capsid confinement on the chromatin organization of the SV40 minichromosome.

Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a 'molten droplet' state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement.

Can repetitive mechanical motion cause structural damage to axons?

Biological structures have evolved to very efficiently generate, transmit, and withstand mechanical forces. These biological examples have inspired mechanical engineers for centuries and led to the development of critical insights and concepts. However, progress in mechanical engineering also raises new questions about biological structures. The past decades have seen the increasing study of failure of engineered structures due to repetitive loading, and its origin in processes such as materials fatigue. Repetitive loading is also experienced by some neurons, for example in the peripheral nervous system. This perspective, after briefly introducing the engineering concept of mechanical fatigue, aims to discuss the potential effects based on our knowledge of cellular responses to mechanical stresses. A particular focus of our discussion are the effects of mechanical stress on axons and their cytoskeletal structures. Furthermore, we highlight the difficulty of imaging these structures and the promise of new microscopy techniques. The identification of repair mechanisms and paradigms underlying long-term stability is an exciting and emerging topic in biology as well as a potential source of inspiration for engineers.

Robotic end-to-end fusion of microtubules powered by kinesin.

The active assembly of molecules by nanorobots has advanced greatly since "molecular manufacturing"­that is, the use of nanoscale tools to build molecular structures­was proposed. In contrast to a catalyst, which accelerates a reaction by smoothing the potential energy surface along the reaction coordinate, molecular machines expend energy to accelerate a reaction relative to the baseline provided by thermal motion and forces. Here, we design a nanorobotics system to accelerate end-to-end microtubule assembly by using kinesin motors and a circular confining chamber. We show that the mechanical interaction of kinesin-propelled microtubules gliding on a surface with the walls of the confining chamber results in a nonequilibrium distribution of microtubules, which increases the number of end-to-end microtubule fusion events 20-fold compared with microtubules gliding on a plane. In contrast to earlier nanorobots, where a nonequilibrium distribution was built into the initial state and drove the process, our nanorobotic system creates and actively maintains the building blocks in the concentrated state responsible for accelerated assembly through the adenosine triphosphate­fueled generation of force by kinesin-1 motor proteins. This approach can be used in the future to develop biohybrid or bioinspired nanorobots that use molecular machines to access nonequilibrium states and accelerate nanoscale assembly.

Osmotically induced reversible transitions in lipid-DNA mesophases.

We follow the effect of osmotic pressure on isoelectric complexes that self-assemble from mixtures of DNA and mixed neutral and cationic lipids. Using small angle x-ray diffraction and freeze-fracture cryo-electron microscopy, we find that lamellar complexes known to form in aqueous solutions can reversibly transition to hexagonal mesophases under high enough osmotic stress exerted by adding a neutral polymer. Using molecular spacings derived from x-ray diffraction, we estimate the reversible osmotic pressure-volume (Pi-V) work needed to induce this transition. We find that the transition free energy is comparable to the work required to elastically bend lipid layers around DNA. Consistent with this, the required work is significantly lowered by an addition of hexanol, which is known to soften lipid bilayers. Our findings not only help to resolve the free-energy contributions associated with lipid-DNA complex formation, but they also demonstrate the importance that osmotic stress can have to the macromolecular phase geometry in realistic biological environments.

Live cyanobacteria produce photocurrent and hydrogen using both the respiratory and photosynthetic systems.

Oxygenic photosynthetic organisms perform solar energy conversion of water and CO2 to O2 and sugar at a broad range of wavelengths and light intensities. These cells also metabolize sugars using a respiratory system that functionally overlaps the photosynthetic apparatus. In this study, we describe the harvesting of photocurrent used for hydrogen production from live cyanobacteria. A non-harmful gentle physical treatment of the cyanobacterial cells enables light-driven electron transfer by an endogenous mediator to a graphite electrode in a bio-photoelectrochemical cell, without the addition of sacrificial electron donors or acceptors. We show that the photocurrent is derived from photosystem I and that the electrons originate from carbohydrates digested by the respiratory system. Finally, the current is utilized for hydrogen evolution on the cathode at a bias of 0.65 V. Taken together, we present a bio-photoelectrochemical system where live cyanobacteria produce stable photocurrent that can generate hydrogen.

Hybrid bio-photo-electro-chemical cells for solar water splitting.

Photoelectrochemical water splitting uses solar power to decompose water to hydrogen and oxygen. Here we show how the photocatalytic activity of thylakoid membranes leads to overall water splitting in a bio-photo-electro-chemical (BPEC) cell via a simple process. Thylakoids extracted from spinach are introduced into a BPEC cell containing buffer solution with ferricyanide. Upon solar-simulated illumination, water oxidation takes place and electrons are shuttled by the ferri/ferrocyanide redox couple from the thylakoids to a transparent electrode serving as the anode, yielding a photocurrent density of 0.5 mA cm(-2). Hydrogen evolution occurs at the cathode at a bias as low as 0.8 V. A tandem cell comprising the BPEC cell and a Si photovoltaic module achieves overall water splitting with solar to hydrogen efficiency of 0.3%. These results demonstrate the promise of combining natural photosynthetic membranes and man-made photovoltaic cells in order to convert solar power into hydrogen fuel.

Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures.

Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 µA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 µA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 µA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants.