Review

Cite This: Chem. Rev. 2020, 120, 288−309 pubs.acs.org/CR

Synthetic Systems Powered by Biological Molecular Motors Gadiel Saper and Henry Hess* Department of Biomedical Engineering, Columbia University, New York, New York 10027, United States

ABSTRACT: 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.

CONTENTS 9. Outlook 300 Author Information 300 1. Introduction 288 Corresponding Author 300 2. Biomolecular Motors: A Brief Introduction 289 ORCID 300 2.1. Biomolecular Motors 289 Notes 300 2.2. Kinesin-1 and Dynein 289 Biographies 300 2.3. Myosin II 290 Acknowledgments 300 3. Linear Biomolecular Motors Applications 290 References 300 Downloaded via COLUMBIA UNIV on April 6, 2020 at 13:58:36 (UTC). 3.1. Actuators 291 3.2. Sensors 291 3.3. Computation 292 4. Rotary Biomolecular Motor Applications 293 1. INTRODUCTION See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. 5. Optimizing Molecular Motors for Nanodevices: From Biomolecular to Artificial Motors and Motor proteins are biological molecular motors responsible for energy conversion and movement from the molecular to the Applications 293 1−5 5.1. Improving the Quality of Biomolecular macroscopic scale in many organisms. The F1-ATPase is Motor Preparations 293 used in biology as a generator, where the rotary motion of the stalk subunit driven by the F0 portion of the F0F1-ATPase 5.2. Hybrid Motors Built with Artificial and 6,7 Biomolecular Motors Components 294 complex is used to produce ATP, but the hydrolysis of ATP 5.3. Artificial Motors 294 can also drive rotary motion. DNA polymerase and RNA 6. Collective Effects and Their Applications 295 polymerase replicate DNA and transcribe genes by generating linear movement.8,9 Myosins use the hydrolysis of ATP to 6.1. Collective Motion of Filaments 295 fi 6.2. Dynamic Self-Organization of Biomolecular exert forces on actin laments and are involved in many cellular processes, including muscle contraction, cell division, Motors 296 ffi 10,11 6.3. Macroscopic Motion Driven by Molecular cargo tra cking, and cell signaling. Kinesins and dynein Motors 297 7. Control of Motion Driven by Biomolecular Special Issue: Molecular Motors Motors 297 Received: April 22, 2019 8. Limitations and Challenges 298 Published: September 11, 2019

© 2019 American Chemical Society 288 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review hydrolyze ATP to “walk” along microtubules and participate in possible ways to overcome them. We will conclude with our − intracellular cargo transport and cell division.12 14 outlook on the future of the field. Our understanding of motor proteins is highly detailed due to their large biological and medical significance which has 2. BIOMOLECULAR MOTORS: A BRIEF engendered an extensive research effort from the biomedical INTRODUCTION ff community. For example, their structures in di erent 2.1. Biomolecular Motors conformational states have been determined with X-ray 15−23 Biomolecular motors are proteins that are responsible for crystallography, their biological roles were elucidated 2,4,5 with biochemical and biophysical methods,24,25 and the mechanical movement in biology. Motors that generate development of single molecule methods enabled new insights linear movement include members of the kinesin and myosin into the coupling of mechanical and chemical events during the protein families, dynein, DNA polymerase, and RNA polymer- − operation of the motor.26 30 This detailed understanding ase, and are for example responsible for muscle contraction and inspired scientists and engineers at the end of the last fast anterograde transport in neurons (Figure 1). Motors that millennium to utilize motor proteins as “off-the-shelf” components in hybrid nanosystems and devise unique applications utilizing these biological molecular motors as − force-producing units.31 35 We have reviewed the progress over the first decade of this effort in 2009,36 and we now aim to provide an update of the progress in the field in the past decade. Three fundamentally different approaches aim to produce active movement in engineered nanosystems: active nano- and microelectromechanical systems (NEMS/MEMS), artificial molecular motors, and biological molecular motors. NEMS/ MEMS-based motors convert electricity to mechanical work.37 They are built from artificial materials, such as silicon, polymers, or carbon nanotubes, and have in principle advantages with regard to stability over protein-based − structures.38 40 However, these motors are generally larger, less efficient, require a dry environment to prevent stiction, and are less compatible with medical applications than biomo- lecular motors. Artificial molecular motors are produced using the methods of synthetic organic chemistry and use ingenious molecular mechanisms to convert light or chemical energy to mechanical work.41 These motors have made tremendous Figure 1. Schematic illustrations of biomolecular motors. (a) DNA polymerase synthesizing DNA. (b) The F0F1-ATPase complex in a progress in recent years, recognized by the 2016 Nobel Prize in membrane, that utilizes the chemical potential of proton gradient to Chemistry to Feringa, Sauvage, and Stoddart,42 and have been 43 convert ADP to ATP, contains the rotary motor F1-ATPase. (c) In used to fabricate contractile polymer gels as well as drill holes muscle, thick filaments assemble from myosin II motors and move 44 into cell membranes to facilitate drug delivery. However, along actin filaments, where troponin together with tropomyosin these motors are synthesized in milligram quantities at the regulates access to binding sites for the motors. (d) Kinesin and laboratory scale and a scale-up of the complex synthesis is dynein move along microtubules, for example, within axons of rather challenging. Biological molecular motors, or biomolec- neurons. Adapted with permission from ref 34. Copyright 2018 ular motors for short, are optimized by billions of years of American Chemical Society. evolution and achieve energy conversion efficiencies of over 40%.45,46 As proteins they are good candidates for biomedical 47,48 can generate rotary movement include F1-ATPase, which is applications and are easy to produce in bacteria and cells. “ ” Furthermore, because biology can be considered to be a proof used in cells as a generator where the rotary movement of the of the feasibility of nanotechnology,49 the study of central stalk unit driven by the F0 subunit is used to catalyze the synthesis of ATP,52 and the flagellar motor, which is biomolecular motors provides both inspiration and a tool 53 chest.50,51 responsible for the movement of bacteria and sperm. Most of Here, we briefly introduce biomolecular motors and then the engineering applications use the linear motors kinesin-1 review the efforts over the past decade to utilize these motors and myosin II, and occasionally dynein. Therefore, we will in engineered systems and devices, including biosensors, focus on these motors. Polymerases generate forces, but the complexity of the actuators, biocomputers, switches, logic gates, screen pixels, ffi 54 and active matter. We will discuss efforts to improve the process make it di cult to exploit in an engineering context. ff Rotary movement driven by F1-ATPase has attracted great available biomolecular motors and also e orts to design new 55 motors incorporating biological building blocks, such as DNA attention in the past, but less work has been done in the past decade. The flagellar motor is difficult to isolate, but hybrid strands or enzymes. We review the work aiming at inducing fl and utilizing collective behavior between motile systems devices incorporating entire bacteria will be brie y discussed below. powered by biomolecular motors, and the work advancing external control over the motile structures powered by these 2.2. Kinesin-1 and Dynein motors. Finally, we will discuss the theoretical and practical The kinesins are a family of biomolecular motors found in limitations of biomolecular motor-powered systems, and eukaryotes that are involved in mitosis, meiosis, and active

289 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review transport in the cell.36 The kinesins’ specific structures and distribution,79 but it can be altered by polymerization − biological functions varies widely between the different motors conditions and further manipulated post polymerization.80 82 in the family56 and provide in principle a wide variety of Microtubules with a protofilament number other than 13 choices to the engineer of hybrid systems. Nevertheless, exhibit a “supertwist”, where the protofilaments wind around kinesin-1 (conventional kinesin) whose primary role is to the microtubule axis with a periodicity of a few micrometers.2 facilitate the transport of molecular cargo along microtubules This supertwist engenders rotation around the axis when from the center of the cell to the periphery has been exclusively microtubules are propelled by surface-adhered kinesins.83 used. Kinesin-1 is a tetrameric protein consisting of two heavy Altogether, microtubules are complex and fascinating nano- chains (binding to microtubules) and two light chains (binding structures in their own right.84,85 57 to cellular cargo). The gene for the heavy chain from 2.3. Myosin II Drosophila has been cloned, and a His-tag has been added to facilitate purification after expression in bacterial or insect Myosins comprise a family of linear biomolecular motors that 48 are involved in organelle transport and cell movement, and cells. Two heavy chains combine to form a functional motor 17,86−90 (often referred to as full-length kinesin) by forming two “head” myosin II is responsible for muscle contraction. Myosin II91 interacts with actin filaments and contains two heavy subunits that bind to microtubules and hydrolyze ATP, a neck- 92 linker region facilitating the conformational change during chains and four light chains. Similar to kinesin, myosin stepping, and an almost 50 nm long tail subunit that terminates converts ATP to mechanical work and in vitro can generate forces of up to 9 pN and velocities of 15 μm/s as it moves in the His-tag and can be used to tether the motor to a surface fi 2,93−96 or artificial cargo. The heads walk toward the plus end of a along actin laments. Myosin II is nonprocessive and 58 detaches after each powerstroke from the actin filament, while microtubule in a spinning hand-over-hand movement. The fi 97 kinesin step size is 8 nm, and it can take more than 100 steps other myosins move processively along the lament. per second at an energy conversion efficiency of over Engineered systems typically use only the larger fragments of 40%.2,45,59 Heavier loads slow the stepping and the motion the heavy chains of myosin II obtained after limited proteolytic stalls at a force of 7 pN.60 Kinesin-1 is a highly processive cleavage with chymotrypsin, termed heavy meromyosin (HMM).98,99 To maintain continuous attachment between motor, which always has one head bound to the microtubule as fi it steps and can perform 100−200 steps on the microtubule the surface/cargo and the actin laments, the nonprocessive ff 61 myosin II motors need to operate in teams. before falling o due to a misstep. Genetic engineering fi permits the truncation of the kinesin tail to a varying degree as Actin, the cytoskeletal lament associated with myosin, self- assembles from globular-actin monomers to form double- well as the creation of a GFP-kinesin fusion protein, which can fi 100 be observed by fluorescence microscopy.62,63 Other motors stranded helical laments with a diameter of 8 nm. Actin from the kinesin family and also from other species may offer provides structure and mechanical strength to the cells as well as connects the transmembrane proteins to cytoplasmic distinct advantages; for example, kinesin-3 from Thermomyces 101,102 lanuginosus has high speed and temperature stability.64 proteins and generates motion in the cell. An actin filament with a persistence length on the order of 10 μmis Dynein is moving about 10-fold slower compared to kinesin- fl 1 motors but in the opposite direction along micro- more exible than a microtubule with a persistence length on 65−70 the order of 1 mm, hence, is more attractive for applications tubules. Axonemal dynein is found in eukaryotic cilia 103−105 65,69 where flexibility is required. and flagella and is involved in their movement. Cytoplasmic dynein is found in animal cells and, similar to kinesin, is involved in cell mitosis and cargo shuttling.69 3. LINEAR BIOMOLECULAR MOTORS APPLICATIONS Dynein has a step size of 8 nm and a stall force of 7 pN and Linear motion is required in many applications and is directly moves at velocities of up to 133 nm/s.67,70 For engineering generated by kinesins, myosins, and dynein.34 In particular the − applications, dynein was, for example, isolated from inverted motility assay or gliding assay,106 108 where the Chlamydomonas reinhardtii.71 biomolecular motors (kinesin-1, dynein or myosin II) are The kinesins and dynein exclusively move along micro- adhered to a glass surface and propel cytoskeletal filaments tubules, tubular structures self-assembling from α,β-tubulin (microtubules or actin respectively), enables the sustained heterodimers with an outer diameter of 25 nm and a length of movement of the filaments over large distances (up to typically several micrometers.72 During the self-assembly centimeters109,110) in contrast to the natural geometry where process, the tubulin heterodimers bind end-to-end to form a cargo is functionalized with kinesin and moves along stationary linear protofilament, and 11−18 protofilaments assemble to microtubules.111 The gliding velocity is governed by the ATP form the microtubule which remains in a dynamic equilibrium concentration and temperature,112,113 and the direction of the between assembly and disassembly.73 Microtubules can be filament movement follows a persistent random walk driven by polymerized in vitro from commercially available lyophilized thermal fluctuations of the tip of the moving filaments.114 This tubulin isolated from bovine or porcine brain. Microtubules persistent random walk, as well as the interaction of the disassemble at low temperatures or if the tubulin concentration filament with obstacles, can be simulated using Brownian − is low, but stabilization with paclitaxel (taxol), or with dynamics methods.115 120 Gliding filaments can be confined − osmolytes as recently discovered,74 can protect microtubules by guiding structures to prescribed paths,36,121 135 directed by − over days against disassembly. Covalent cross-linking with obstacles,136 controlled by light,36,137 146 or other stimu- − − bifunctional cross-linkers can extend the microtubule lifetime li,36,147 152 and can capture analytes and carry cargo.36,153 163 for a week.75 Fluorescently labeled and biotinylated tubulins The natural arrangement of stationary filaments supporting the are also commercially available, and several new strategies to movement of biomolecular motors is also employed, but the conjugate linkers and cargo to microtubules have been transport distance is largely limited to the length of the − described.76 78 The in vitro microtubule length distribution filament or filament assembly in this geometry. These has been found to be best described by a modified Schulz discoveries led to more advanced applications including

290 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review

− sensors,164 167 computation,168 screens,71 and switches.169 In electric field, the microtubules are superior actuators in this this section, we will highlight several applications for linear application compared to the tip of an atomic force microscope. biomolecular motors and filaments as well as a few using Kinesin-propelled microtubules have been previously used for applications with rotary motors. topographical mapping,182,183 but the work of Gross et al. 3.1. Actuators added the mapping of electric fields and the work of Inoue et al. demonstrated the mapping of surface strain.167,184 An Actuation is a key role of linear biomolecular motors in fi biological systems, both on a macroscopic scale in muscles5 alternative con guration is the use of immobilized micro- 170 tubules in ordered arrays (e.g., created by pick-and-place and on a microscopic scale within cells. In engineered 185 systems, biomolecular motors have been shown to shuttle assembly ), supporting the bidirectional transport and − 186 cargo36 ranging in size from molecules171 176 over nano- and assembly of cargo by kinesins and myosins. − fi microparticles177 179 to cells.180 An illustrative application is A long-term goal in the eld is to scale-up molecular force generation and mimic the hierarchic organization of found in the work of Gross et al., who used kinesin propelled 187−189 microtubules carrying quantum dots to map the electric field muscle. Progress has been made in the construction fi distribution on a surface patterned with nanogold slits (Figure of arti cial cilia, where microtubules are cross-linked by kinesin 2).181 Because of their small size and weak interaction with the constructs and the resulting microtubule bundles exhibit beating movement190,191 as well as in the construction of contractile networks of motors, filaments, and in some cases − microparticles.192 194 However, the creation of ordered arrays of filaments which are actuated by large numbers of motors and then sandwiched into multilayer structures as shown in Figure 3 is still in the conceptual stage.195 The work of Agayan et al. highlighted how thermal forces oppose the dynamic assembly of ordered arrays.196 Nevertheless, such molecular engines could have significant roles in medical application such as prosthetic limbs and hold great promise for the future. 3.2. Sensors Sensors are devices that detect changes in the environment and are used for health care, environmental, and military purposes. Biosensors often use antibodies to bind to biological target molecules and detect this binding with a physical or chemical − detector.197 199 In a double-antibody sandwich biosensor, surface-bound antibodies capture an analyte molecule. Excess analytes are washed out and tagged antibodies are added and Figure 2. Mapping of electric fields with motor-propelled nanoprobes. bind to the analyte. Another wash is needed to remove the (a) Schematic representation of microtubules with attached quantum excess tag-antibody and then bound tags can be detected. A dots propelled by kinesins along a surface with gold nanoslits. The sensor can integrate active transport powered by molecular changes in fluorescence intensity when the quantum dots interact with the electric field are detected with fluorescence microscopy. (b) motors to reduce the time required for analyte capture and Maximum intensity projection of the quantum dots as they are detection and to remove the need for washing steps and the 172,200−204 164 transported across the surface. Gray dash lines mark the gold slits. associated machinery. For example, Fischer et al. Adapted with permission from ref 181. Copyright 2018 Nature. constructed a two-dimensional smart dust biosensor. In this device, antibody-functionalized microtubules capture analytes,

Figure 3. Macroscale engine concepts based on molecular motors. (a) Schematic design of a millimeter size engine utilizing coupling of kinesin motor activity via microtubules, ordered microtubule arrays, and sandwiched layers. (b) A patented rotary motor concept powered by myosins. Drawing taken from patent US 7,349,834.195

291 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review are then propelled by surface-adhered kinesins and encounter where an analyte can be collected, and a trapping zone, where a fluorescent nanoparticles functionalized with a second anti- detector can be used. The zones are coated with myosin and body. After binding several of these tags to the analyte, the connected with a nanochannel. Once ATP is added, actin microtubules were then propelled to a detection area where the filaments in the loading zone are propelled to the trapping arrival of the fluorescent tags indicates the presence of the zone within 60 s. The high concentration of actin in the 10 analyte. Lard et al.205 improved on this design by using myosin μm2 detection area provides a high signal-to-noise ratio. motors and actin filaments to construct an ultrafast biosensor Optical interactions between motile filaments and nano- (Figure 4). This nanoseparator is built with a loading zone, sctructures can further enhance sensing.165,206 Adifferent approach was taken by Kumar et al.174,176 and later Chaudhuri et al.204 to design a biosensor with a label free detection scheme (Figure 5). Multivalent analytes bind to two antibodies attached to different filaments leading to cross- linking and bundling between the filaments when they are propelled by surface-adhered motors. These filament bundles were easily detected using fluorescence microscopy. Overall, a number of interesting demonstrations have been published in the past decade, yet an application promising enough to justify commercial development has not yet been found. This situation is reminiscent of microfluidics, which, despite fewer technical hurdles compared to microanalytic systems integrating biomolecular motors, has struggled to fully deliver on its promise in the commercial arena.207,208 3.3. Computation The use of motile agents for computation has been introduced by Nakagaki et al.209 and Nicolau et al. suggested in 2006 to employ biomolecular motors and filaments.210 This vision was realized in 2016 in a study by an international team168 describing parallel computation using kinesin propelled Figure 4. Biosensor using myosin and actin. (a) Structure of the microtubules or myosin propelled actin traveling within a biosensor (or nanoseparator). The myosins propel actin from the network of channels. The network consisted of two types of loading zones (CON-LZ) to the trapping zone (TZ) via nano- junctions: (1) a pass junction where a filament will continue in channels. Control loading zones (CTR-LZ) are not connected to the the same direction it came from and (2) a split junction where trapping zone with nanochannels. (b) Scanning electron micrograph a filament will decide to move in one of two paths at a 50/50 of a nanochannel. (c,d) Fluorescence images of TRITC-phalloidin probability (Figure 6a). The arrangement of these two junction labeled actin (c) before adding ATP and (d) after adding ATP. The types can encode combinatorial problems, such as the subset actin is propelled from the loading zones to the trapping zone. sum problem for the set {2, 5, 9} (Figure 6b). This device uses Adapted with permission from ref 205. Copyright 2013 Elsevier. 4 orders of magnitude less energy per operation than an electronic computer, due to the low energy consumption by

Figure 5. A label-free biosensor. (a) Schematic diagram showing the stages of detection. Microtubules with antibodies capture analytes and are propelled by kinesin. The analytes can cross-link microtubules and the bundled microtubules can be detected. (b) Fluorescence microscopy images of labeled microtubules before and after adding of an analyte (leukemia microvesicles). Scale bar 10 μm. Adapted with permission from ref 204. Copyright 2017 American Chemical Society.

292 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review

Figure 6. Parallel computation with biomolecular motors and filaments (a) Top: schematic representation of the pass (left) and split junction (right). Bottom: maximum projection images of microtubules passing through a pass junction (left) and a split junction (right). (b) Diagram of the computation network to compute possible solution for the sums of the numbers 2, 5, 9. The filaments enter from the top left corner and go through pass junctions (empty circles), where they continue straight, and split junctions (filled circles), where they will continue straight or turn with equal probability. The exit numbers correspond to possible solutions, coded as correct (green) and incorrect (magenta) results. For example, in the path marked in yellow the filament turns on the first and third split junction and continues straight on the second leading to a solution of 9 + 2 = 11. In the blue path, the filament continues straight in all split junctions leading to the result of 0. Adapted with permission from ref 168. Copyright 2016 U.S. National Academy of Sciences. the propelling motors, however, correcting errors remains a 5. OPTIMIZING MOLECULAR MOTORS FOR challenge168 and the speed of the computation has been NANODEVICES: FROM BIOMOLECULAR TO − debated.211 213 In an alternative approach to biocomputation, ARTIFICIAL MOTORS AND APPLICATIONS DNA strands were used to cross-link DNA-functionalized Biomolecular motors are evolutionarily optimized for nature’s microtubules gliding on surface-adhered kinesins and thereby use; however, they are not optimized for engineering logic gates were implemented where aggregation of micro- applications. To improve the utility of biomolecular motors, tubules depended on the addition of the correct cross-linker.169 a few approaches can be taken. The first approach is to While solving complex mathematical problems may remain a improve the preparation of the biomolecular motors to obtain task for electronic computers, it might be beneficial to process a highly purified and functional protein. The second approach the exponentially increasing amounts of molecular information is to modify biomolecular motors to further improve their generated (e.g., in the form of DNA sequences from patient performance using protein engineering methods and to and environmental samples) with molecular approaches. In combine biomolecular motors with other molecules, such as this context, active transport and force generation by RNA, in order to engineer an improved hybrid motor. Finally, a prominent approach is the design of artificial molecular biomolecular motors may be critical for tasks such as motors from biological building blocks, such as DNA strands, information transfer and proofreading. Furthermore, future but without the use of any components from existing autonomous micro- and nanorobotic systems will require biomolecular motors. These approaches were previously distributed information processing which is integrated with 224,225 84 discussed in several reviews. Similarly, the motor- sensing and actuation. associated filaments can be engineered by covalent and noncovalent modifications.226 Such modifications are ideally 4. ROTARY BIOMOLECULAR MOTOR APPLICATIONS explored in high-throughput fashion227 and analyzed with tracking software specifically adapted to the task of identifying Several applications of rotary motors, in particular of the and characterizing filaments, such as the popular FIESTA flagellar motor, have been described in the past decade, 228 package. including micro/nano power generators, self-powered actua- − 5.1. Improving the Quality of Biomolecular Motor tors, and microswimmers.214 219 Microswimmers, in this Preparations context, are hybrid devices that combine a flagellar motor A key approach in biotechnology to increase protein with a synthetic or biologically based vesicle and aim to mimic fi the movement of bacteria or sperm cells.220 The vesicle can be productivity is to optimize the selection, puri cation, and preparation of the proteins,229 and a similar approach can be used to deliver cargo, such as drugs, to different cells or parts of used to improve biomolecular motors stability and perform- the body. For example, the microswimmers could be used to 64 48 220,221 216 ance. In a recent study, Korten et al. expressed kinesin-1 in target cancer cells or improve fertilization. Adding insect cells rather than bacteria because expression of magnetic components to the microswimmers can increase 222 eukaryotic proteins in bacteria may cause decreased function- propulsion and external controllability. These devices ality due to lack of chaperones and early translation compete with bacteria genetically engineered for similar termination.229,230 The kinesin was expressed in insect cells applications,223 but their inability to multiply may be an and then highly purified. In a gliding assay using these kinesins, important safety feature and advantage. microtubule velocity was stable for over 25 h (Figure 7a).

293 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review

Figure 7. Kinesin-1 expression in insect cells. (a) PAGE of kinesin expressed in bacteria and insect cells shows the increased purity of the Figure 8. Hybrid molecular motor engineered with myosin and RNA. protein from the insect cells. (b) Distribution of the velocity of the (a) Left: schematic representation of the motor. Right: schematic microtubules with kinesin expressed in bacteria (left) and in insect representation of the movement of the motor. (b) Fluorescence cells (right) after 3 h of gliding. Adapted with permission from ref 48. microscopy image showing the movement of actin filaments propelled Copyright 2016 IEEE. by the hybrid motor. (c) Histogram of the velocity of the actin using two different RNA concentrations. Adapted with permission from ref Because of fewer defective kinesins, the microtubules do not 235. Copyright 2018, Nature. break as much and do not leave the surface as frequently. This study showed that altering the preparation of the biomolecular of these engineered motors with unique properties are adopted fi motors can signi cantly enhance the performance of the by engineers engineering devices. 231 fi system. Rahman et al. focused on the puri cation of myosin 5.3. Artificial Motors II solutions in order to reduce the effects of defective motors ff A competing approach to the utilization of biomolecular but found unexpected trade-o s between the smoothness and − fi motors is the design of fully artificial motors.41,236 238 These the velocity of actin laments gliding on the surface-adhered 239−243 244 myosins. motors include DNA walkers and spiders as well as rotaxanes, catenanes, and Feringa-type rotary motors produced 5.2. Hybrid Motors Built with Artificial and Biomolecular 41,238,245−248 Motors Components by organic synthesis. These motors can exceed the performance of biomolecular motors in specific metrics. For The properties and performance of biomolecular motors can example, a rotaxane was shown to generate forces of 30 pN249 also be improved by engineering the motor and adding other 238,250 − and a catenane was shown to produce rotary work. Some macromolecules.139,143,232 234 Several studies from Prof. Zev ’ motors are designed with the explicit goal of testing our Bryant s research group engineered biomolecular motors to mechanistic understanding of force production. For example, change and improve their motor functions. Myosin was Kovacic et al.251 designed a “lawnmower” motor to move on a designed to respond to calcium and change its direction of substrate path. The motor is designed with a quantum dot hub motion.139 Both kinesin and myosin were engineered to speed “ ” 143 bonded to protease blades (Figure 9). Experiments up, slow down, or switch directions when exposed to light. demonstrated that the proteases were active in this In another study, myosin was engineered to not only be 233 construction, and simulations indicated that the lawnmower controlled and bidirectional but to also be more processive. can move along a filament with proteins as binding and This was achieved by designing three and four headed myosin fl cleaving sites for the protease. Alternative designs by the same and by introducing exible elements between the heads. team are the “tumbleweed”, “inchworm”, and “bar-hinge” Finally, a hybrid molecular motor integrating a myosin motor and RNA lever arms was engineered.235 In this hybrid motor, conformational changes in the myosin were amplified by the RNA arm to generate a larger stroke. The motor has the ability to move filaments over 2 μm at speeds of ∼20 nm/s (Figure 8b). They used several unique RNA lever sequences, which allowed for bidirectional processive motion and for controlled switching between directions. Furuta et al.234 created a novel motor by combining the dynein motor core with actin binding fi proteins. This motor moved actin laments in a gliding assay in Figure 9. Schematic representation of the lawnmower molecular a direction determined by the assembly of building blocks. motor. The protease blades (red, e.g., trypsin) are linked to the Such engineered motors can be used for bidirectional transport quantum dot hub (green circle). The protease binds and cleaves systems, which, unlike kinesin or dynein alone, can transfer DNA-bound peptides. Adapted with permission from ref 251. cargo in both directions. It will be fascinating to see which one Copyright 2015 IEEE.

294 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review

Figure 10. DNAzyme-based molecular motor. (a) Schematic illustration of the DNAzyme-based molecular motor carrying a CdS nanocrystal (yellow) and walking on an RNA-decorated nanotube track (black). The DNAzyme catalytic core (E) motor binds to the RNA (S1) and cleaves it (pink) in the presence of Mg2+ to short (P1) and long (P2) fragments. P1 then dissociates from E and the unbound arm of E associates with an adjacent RNA (S2). The rest of the E moves from S1 to S2, completing the step and returning to the initial conformation. (b) Distance covered by the DNAzyme motor as a function of time. The motor moves only in the presence of Mg2+. Adapted with permission from ref 257. Copyright 2014 Nature.

− motors.252 255 A similar burnt-bridges motor256 relies on a solution induce bundling in a concentration dependent manner DNAzyme, that is a RNA-cleaving DNA strand, to transport (Figure 11).269,286 nanocrystals along a RNA decorated carbon nanotube (Figure 10).257 This motor walked along the nanotube track processively and autonomously, similar to biological molecular motors. Interestingly, some researchers tried to produce force and directed motion by exploiting external gradients258,259 and randomly self-generated gradients.260 We believe that, as a result of the continuous technological improvements in our ability to design and fabricate proteins driven by the pharmaceutical industry, biomolecular motors adapted by protein engineering will emerge as the primary technological route to convert chemical energy into mechan- ical work. 6. COLLECTIVE EFFECTS AND THEIR APPLICATIONS The past decade has seen an increasing focus on collective motion of biomolecular motor-propelled filaments, building on the work utilizing individual filaments36,261 as well as active self-assembly.262,263 This focus is often inspired by swarming, a behavioral state where swarms of bees, flocks of birds, and schools of fish aggregate and move collectively in a synchronized pattern. In this section, we discuss the Figure 11. fi Swarming induced by depletion forces. (a) Schematic interactions between laments leading to swarming and self- diagram of the gliding assay. Microtubules are propelled by surface- assembly, the fascinating phase transitions arising in these adhered kinesins. Adding methylcellulose (MC) induces depletion systems, and the possible applications arising from this forces between the microtubules. (b) Florescence microscopy images behavior. of microtubules without MC (left) with 0.1 wt % MC (center) and 6.1. Collective Motion of Filaments 0.3 wt % MC (right). The depletion forces induced by the MC cause the formation of microtubule bundles. Reproduced with permission Collective motion of filaments arises from the interplay of from ref 269. Copyright 2015 Royal Society of Chemistry. attractive and dispersive forces, and its theoretical description requires advances in the science of active matter and its phase − transitions.264 267 Swarms of filaments can exhibit self- assembly, bundling, and collective movement in response to Programmable interactions of microtubules can be used to confinement, induced forces, and engineered interac- engineer logic gates and switches.169 For example, Keya et − tions.169,190,268 272 Steric interactions between filaments can al.169,272 chemically attached single-stranded DNA (ssDNA) to be tuned by varying the filament length and density, typically microtubules (MT-DNA) and performed gliding assays. Once resulting in a transition from an isotropic phase to a nematic complementary ssDNA strands (l-DNA) were added to the phase.268,273,274 Importantly, the active motion introduces solution, the l-DNA linked the MT-DNA on the microtubules features which are absent in passive equilibrium systems, such together, which caused bundling and collective motion (Figure as coexistence of nematic and polar states.275 Furthermore, 12a,b). To “switch off” the swarming, additional ssDNA (d- interactions with the boundaries affect the system.270 Strong DNA)wasaddedtobindtothel-DNAviastrand interactions between filaments, induced, e.g., by cross-linking displacement and thereby remove the cross-links. Switching of biotinylated microtubules by streptavidin178,276 or actin by light can be achieved by azobenzene-modified DNA strands, filaments by fascin,277 lead to the formation of long-lived wire- which transition between cis and trans conformations under − like and ring-like structures.263,276,278 285 Weak interactions illumination (Figure 12c,d). Visible light induces the trans between filaments can result for example from depletion forces, conformation exposing the DNA and causing the bundling of where macromolecules such as methylcellulose added to the the microtubules. UV light induces the cis conformation,

295 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review

Figure 12. Microtubule swarming controlled by DNA. (a) Schematic representation of association and dissociation of DNA-functionalized microtubules by adding the complementary DNA strand. (b) Fluorescence microscopy images of the microtubules gliding on kinesin-coated surfaces at t = 0 (left), when the complementary DNA strand was added, and t = 4200 s (right). (c) Schematic representation of a light-controlled switch. Visible light causes the azobenzene to change to a trans conformation, allowing association between the azobenzene−microtubule complexes. UV light induces the cis conformation of the azobenzene groups, which disrupts DNA interactions and causes unbundling. (d) Fluorescence microscopy images of the microtubules under UV light (left) and under visible light (right). Adapted with permission from ref 169. Copyright 2018 Nature.

Figure 13. Dynamic recruitment of kinesin-1 motors fused to green fluorescent protein (GFP) to a weakly binding surface by microtubules. (a) Schematic representation of a microtubule propelled by GFP-kinesins (green) that are recruited from the solution by the microtubule, attach to the surface, are left behind, and detach from the surface within minutes. (b) Fluorescence microscopy image of the microtubules (red) and the kinesin motors (green). (c) Fluorescence time-lapse images of kinesin being deposited and then released within 2 min after the microtubule passes. Left: 647 nm (red) channel showing the microtubules. Center: 488 nm (green) channel showing the kinesin motors. Right: overlay of both microtubules and kinesin. Adapted with permission from ref 292. Copyright 2018 American Chemical Society.

“hiding” the DNA, and leading to unbundling of the transition as a function of the grafting density,287 the myosin microtubules. motor conformation has been shown to be sensitive to the 6.2. Dynamic Self-Organization of Biomolecular Motors surface properties.288,289 Kinesins and myosins can be Dynamic and collective behavior can also be observed at the anchored in lipid membranes, where they retain mobility and organizational level of the biomolecular motors. While steric can dynamically interact with each other through forces 290,291 292 interactions between surface-adhered kinesins can affect their transmitted over filaments. Lam et al. performed conformation and, for example, cause a layer of surface- experiments where the kinesin is not permanently bound to the adhered full-length kinesins to undergo a mushroom-to-brush surface but is able to detach from and reattach to a surface

296 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review coated with Pluronic-F108 copolymer functionalized with Ni- NTA (Figure 13a). In this setup, microtubules can bind kinesins from solution, use them for propulsion, and leave them behind in a slowly vanishing trail (Figure 13b,c). This system achieves for the first time dynamic turnover of motor building blocks in an artificial system, a property which enables for example heart muscles to sustain their operation over decades.293 Efforts to induce synchronization between permanently surface-adhered kinesin motors attached to the same micro- tubule by increasing their density were unsuccessful in the sense that a characteristic increase in velocity fluctuations could not be observed.294 The velocity fluctuations were instead shown to be consistent with a model by Sekimoto and Tawada295 and likely arise from the variability in the kinesin force generation as a result of their variable attachment geometry. Recent work pushes toward better answers to fundamental questions, such as: how many motors are on the surface, how Figure 14. An active gel that contracts upon illumination. (a) many motors are attached to a filament, and what is the Illustration of the contraction of the cross-linked polymer upon activation of the rotary motor units by illumination. The open collective force generated? Fallesen et al. inferred motor- fi spacing along a microtubule and collective forces of a few con guration (left) coils up and decreases its volume (right). (b) Snapshots of the gel before illumination (left) and t = 120 min after motors by pulling a magnetic bead,296,297 while VanDelinder et fl illumination (right). Adapted with permission from ref 43. Copyright al. employed uorescence interference contrast microscopy 2015, Nature. (previously used to determine the distance between a kinesin- propelled microtubule and the surface298) to determine the relation between kinesin surface density and kinesin spacing on Many applications require all the filaments to be aligned to a microtubule.299 DNA nanotechnology enables the coupling achieve a synchronized motion that can exert a microscopic of precise numbers and types of motors with defined spacing force. Control over the direction of the filaments has been − 91,196,312 313 and provides insight into motor coordination.300 302 Multiple achieved by flow, electric fields, magnetic 314−316 317 surface-adhered kinesins were found to be capable of fields, and by engineering channels for the filaments. generating translational forces of up to 100 pN on a For example, Lindberg et al. designed specific channels for microtubule,303 but it is still rather unclear which forces can actin filaments to move in by coating the surface and achieving 99 be generated at high motor densities. different surface hydrophobicities. Another important goal is the ability to control the velocity and start and stop the 6.3. Macroscopic Motion Driven by Molecular Motors movement of the filaments.143 Some studies used light to The amplification of the motion generated by individual release caged ATP to provide fuel to the biomolecular molecular motors into macroscopic motion is elegantly motors,137,138 while others used light to release a caged achieved in muscle tissue304 and a goal for synthetic and inhibitor and slow the motors.318 Azo-peptides have been hybrid systems.194,305 Torisawa et al. succeeded in creating synthesized which can act as light-switchable inhibitors for − millimeter-scale contractile networks of microtubules and the kinesin and permit localized activation.319 321 Myosin II motor homotetrameric kinesin Eg5.193 Even more striking is the work activity has been switched by altering the oxidative status of the of Li et al.,43 who engineered active matter in the form of a environment,322 which putatively is related to alterations to the polymer gel that contracted upon light illumination (Figure regulatory light chains.323 14). They used light-driven artificial rotary motors306,307 and Different control strategies can be combined to achieve self- designed polymer−motor conjugates. Upon illumination, the organization.71,324 Aoyama et al.71 combined self-assembly, motor rotated, twisted the polymer, and decreased the volume microtubule organization, and light-controlled switching to of the gel. Interestingly, although the mechanism for engineer a molecular motor-based melanophore or “pixel”. contraction is entirely novel, the generated stress is on the They attached microtubule seeds to the center of a hexagonal order of 100 kPa, similar to the stress generated by muscle308 chamber and created polar asters by extending the seeds in a and adhering closely to the universal limit for motors identified polymerization step (Figure 15a,b). Then they added dynein by Marden and Allen309,310 and previously discussed by us.32,34 attached to fragments of fluorescently labeled microtubules, which were evenly distributed along the elongated micro- 7. CONTROL OF MOTION DRIVEN BY tubules and stationary in the absence of ATP (Figure 15c). UV BIOMOLECULAR MOTORS light released caged ATP causing the dyneins with the fluorescent microtubule fragments to travel to the center of For applications it is critical to control biomolecular motors the chamber. This concentration of the fluorescently labeled and filaments with respect to their direction and velocity of microtubules in the center rendered a large area of the movement, their force generation, and alignment both chamber free of fluorescent material, and thus the chamber temporally and spatially. For example, in future nanoengines, appeared now to be dark (Figure 15d). The authors arranged the filaments need to be aligned and the motors need to be 7500 of these “artificial melanophores” in a 4 mm × 4mm powered on and off.261,311 Here we focus on the progress over array and illuminated them with UV light through a photomask the past decade, for prior work, see, e.g., ref 36. (Figure 15e). Only in the illuminated melanophores did the

297 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review

Figure 15. Biomolecular motor-based optical device. (a) Microtubule seed polymerizing outward. (b) Fluorescence microscopy image of a radial microtubule array in a chamber. (c) Illustration of the transport of the fluorescently labeled microtubule fragment by the dynein to the microtubule minus end in the center of the chamber. (d) Fluorescence microscopy images of the microtubule fragments at UV illumination (left) and 8.5 s after UV illumination (right). The UV light releases caged ATP, which induces the movement of the dynein to the center of the chamber. (e) Schematic representation of a photomask, where UV light passes through the pattern and allows the ATP to be released only in specific melanophores. (f) A screen composed of thousands of artificial melanophore chambers each acting as a pixel with two color options. By using the photomask a pattern can be created on the screen. Adapted with permission from ref 71. Copyright 2013 U.S. National Academy of Sciences.

Figure 16. Wear of kinesin-propelled microtubules. (a,b) Microtubule wear measurements using fluorescence microscopy. (a) Plot showing the length of a microtubule as a function of time. (b) Histogram of the shrinking rate of microtubules for stationary and moving microtubules. (a,b) Adapted with permission from ref 339. Copyright 2015 Nature. (c) High speed atomic force microscopy images of microtubules showing the microtubule gliding and splitting off of a protofilament (PF). (d) A schematic drawing of a model showing the breaking of the protofilament (from c) after a sudden change of direction. (c,d) Adapted with permission from ref 341. Copyright 2017 Nature. pigmentation change, which allowed the formation of a pattern replace biomolecular motors, for example, myosin is replaced (Figure 15f). in muscle tissue every few days293,325 and kinesin-3 from Caenorhabditis elegans is removed once it loses its specific 8. LIMITATIONS AND CHALLENGES binding to cargo.326 While mechanical stresses may play a The current applications of biomolecular motors are struggling significant role in the case of molecular machines such as with problems related to lifetime, scale-up, and cost. In this biomolecular motors,327 oxidative stress is a general reason for section, we will discuss these problems and try to understand if protein degradation.328 The importance of the removal of they are theoretical limits or if engineering solutions can be reactive oxygen species (ROS) has been demonstrated by found. Kabir et al. when they placed their gliding assay in a nitrogen- The functioning of the above-described devices and systems filled chamber to remove the oxygen in the solution and is limited by the lifetime of the biomolecular motors and the extended the lifetime of the kinesin motors from a few hours to stability of the filaments. In biology, the cells continuously a few days.329 These insights were confirmed by related work

298 DOI: 10.1021/acs.chemrev.9b00249 Chem. Rev. 2020, 120, 288−309 Chemical Reviews Review from other groups.330,331 The ROS-free environment also Wear, breaking, and mechanical fatigue are all engineering enhanced the dynamicity of microtubule polymerization.332 effects that affect the lifetime of biomolecular motors and Label-free imaging, e.g., via interference reflection micros- filaments. The study of these effects can lead to improved and copy,333 may reduce the need for high intensity illumination. longer lasting devices and at the same time illuminate the Microtubules and actin filaments are intrinsically dynamic fundamental design principles guiding the evolution of assemblies and are typically stabilized against depolymerization biological structures.346,347 by paclitaxel (taxol) or phalloidin for a day or two. The lifetime However, the operation of biomolecular motors and their of microtubules can be extended to a week by cross-linking of associated filaments in environments other than their functional groups on the tubulin surfaces.75 Recently, it has optimized buffer can introduce additional challenges. While been shown that microtubule depolymerization is not only Bachand and Bachand found that several common environ- inhibited by small molecules, such as paclitaxel,334 but also by mental contaminants are well tolerated by kinesins and the presence of osmolytes in solution.74 The limited supply of microtubules,348 Korten et al. demonstrated that specifically ATP can be potentially extended indefinitely by the integration blood, plasma, and serum even at hundred-fold dilution of photosynthetic systems.335 interfere with the operation of kinesin- and myosin-driven Many other biohybrid systems have a similar struggle with systems.349 Kumar et al. solved this problem by capturing lifetime, for example, photosynthesis based solar cells have a analytes from serum, exchanging the buffer, and then very short lifetime compared to photovoltaic solar cells.336,337 introducing the motors.350 Clearly, protein engineering to Constant replacement of the protein components is the mitigate adverse effects, protected environments351 or effective biological solution to this problem, and section 6.2. describes packaging solutions352 are needed. the first steps toward engineered systems with continuous The challenges in scale-up can refer either to the cycling of building blocks. However, certain applications may magnification of the force output to the macroscale illustrated not require an active lifetime exceeding a few minutes or in Figure 3, or the mass production of small devices, such as hours.201,203 the “smart dust” biosensor described in section 3.2. Both Mechanical activity leads to degradation (wear and fatigue) require larger amounts of motor proteins as well as suitable at the macroscale and should also contribute to degradation in solid-state fabrication techniques and high-speed liquid nanosystems powered by molecular motors. For example, handling procedures. The packaging and storage of large filaments can break and undergo wear, defined as a gradual loss assemblies of fragile proteins, such as kinesin and tubulin, also of material from a body.338 Dumont et al.339 measured the presents new challenges, although early attempts at lyophilizing wear of the taxol-stabilized microtubules and showed that the or simply freezing motor proteins and filaments in devices were microtubules get shorter over time (Figure 16a). The shrinking surprisingly successful.353,354 The continued advances in the rate for kinesin propelled microtubules was higher than for production of biologics in the pharmaceutical industry will stationary microtubules (Figure 16b). Furthermore, increasing facilitate a potential scale-up in the production of biomolecular the kinesin density or increasing the velocity increased the motor-powered devices by supporting a technological environ- shrinking rate. Reuther et al. highlighted that at low kinesin ment devoted to the manufacturing, packaging, and storage of surface densities, under 20 μm−2, the shrinking rate of active proteins. The resulting progress in biotechnology will stationary microtubules decreased with increasing density.340 drive down the costs for protein engineering, protein This suggests that the attachment to the surface via kinesins expression, and protein purification. At the same time, protects the microtubules from depolymerization to a certain biomolecular motors and the related devices and materials degree and indicates the need for further investigations. Keya can serve as a testbed for the process engineer because they et al.341 made striking direct observations of the wear process represent particularly challenging problems. of kinesin-propelled microtubules using high speed atomic Thecostofemergingtechnologyisalwaysinitially force microscopy. The high resolution of the imaging astronomical and then is reduced dramatically as adoption technique allows the observation of individual microtubule broadens. With respect to molecular motors, we are at the very protofilaments (Figure 16c). Their results indicated that beginning of this process, where, for example, myosin II protofilaments break off at the leading edge of the gliding motors are commercially available at a price of $40US/mg. For microtubule. Moreover, they showed that the split protofila- comparison, milk powder sells for $4US/kg, which is a price 10 ment is still propelled by kinesins but at a slower velocity than million times lower and not far from commodity materials such the mother microtubule. The authors suggested that an as polyethylene and steel. The history of solar cells is an overhanging protofilament at the leading edge of the excellent example of the technology adoption process and microtubule bound to a defective kinesin leading to a illustrates the need for government support at all stages of directional change and breaking of the protofilament (Figure technology development. However, just as solar cells found 16d). This adds detail to the prior observations of VanDelinder their initial niche in spacecraft, we will have to identify a high et al., who observed splitting of gliding microtubules into value application for molecular motor driven devices. protofilament bundles.342 A challenge which seemed answered after extensive research In addition to shrinking of microtubules from the ends, in the past three decades is the development of a mechanistic internal tubulin removal and ultimately breaking of micro- understanding of molecular motor design. Theoretical and tubules is also a possibility. Schaedel et al.343 performed computational work clarified the relevant principles of physics bending experiments on microtubules via cycles of bending and chemistry for both biological and synthetic molecular − and release. These measurements indicated that microtubules motors,355 357 although surprises such as a revision of the stiffness decreased with every cycle and that the filaments kinesin stepping mechanism still emerge.58 Unfortunately, here underwent materials fatigue. They further showed that free we can only cite a few of the numerous contributions. tubulin rebuilt the microtubule, allowing the microtubule to However, recent reports regarding “enzyme propul- − regain its original strength.344,345 sion”,13,358 361 where enzymes are found to exhibit increased

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Myosins: Domain Altogether, molecular motors are poised to serve in key roles in Organisation, Motor Properties, Physiological Roles and Cellular materials science, active matter, and synthetic biology research. Functions. In The Actin Cytoskeleton: Handbook of Experimental Pharmacology; Jockusch, B. M., Ed.; Springer: Cham, 2016; Vol. 235, AUTHOR INFORMATION pp 77−122. Corresponding Author (12) Hirokawa, N.; Noda, Y.; Tanaka, Y.; Niwa, S. Kinesin Superfamily Motor Proteins and Intracellular Transport. Nat. Rev. *Phone: 212-854-7749. E-mail: [email protected]. Mol. Cell Biol. 2009, 10, 682−696. ORCID (13) Riedel, C.; Gabizon, R.; Wilson, C. A. M.; Hamadani, K.; Tsekouras, K.; Marqusee, S.; Presse, S.; Bustamante, C. The Heat Gadiel Saper: 0000-0002-3556-7977 Released during Catalytic Turnover Enhances the Diffusion of an Henry Hess: 0000-0002-5617-606X Enzyme. Nature 2015, 517, 227−230. Notes (14) Reck-Peterson, S. L.; Redwine, W. B.; Vale, R. D.; Carter, A. P. 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