Biomolecular Motors Research Articles

Bionanotechnology: its applications and relevance to healthcare

The application of nanotechnology in the field of biology and medical sciences is known as bionanotechnology. This technology gives rise to a new device and system with greater sensitivity and accuracy. The important applications in this field of study include synthesis of new molecules, selfless assembly of structures from DNA, macromolecular science and engineering mimics biological assembly, drug-delivery systems, therapeutic applications, biomolecular motors, bioelectronics, DNA computers, enabling technologies, etc. Finally, we will also take a look at the future of bionanotechnology with respect to US dollars spent on it.

Shape Control of Filamentous Motor Proteins for Bio-Nano Driving Units

This paper describes a technique for controlling the shape of filamentous motor proteins for bio-nano driving units in MEMS devices. In this experiment, we have used actin, a protein to construct cytoskeleton. Actin monomers (G-actin) polymerize in high salt condition and form filaments (F-actin); the filaments move when they bind with the motor protein (myosin) in ATP (adenosine tri-phosphate) solution. Fascin, a putative bundling protein, tightly bundles several F-actins together to form tight bundles of actin and polymerized in the polydimethylsiloxane (PDMS) or parylene micro chambers, and then formed several shapes, such as circles, rods, triangles or squares. Since the bundled actins still have motility, we believe this technique is useful for forming a desired pattern of bio-molecular motors toward the actuation of MEMS/NEMS devices.

Recent developments of bio-molecular motors as on-chip devices using single molecule techniques

The integration of microfluidic devices with single molecule motor detection techniques allows chip based devices to reach sensitivity levels previously unattainable.

Biomolecular motor-driven microtubule translocation in the presence of shear flow: analysisof redirection behaviours

We suggest a concept for powering microfluidic devices with biomolecular motors andmicrotubules to meet the demands for highly efficient microfluidic devices. However, tosuccessfully implement such devices, we require methods for active control over thedirection of microtubule translocation. While most previous work has employed largelymicrofabricated passive mechanical patterns designed to guide the direction ofmicrotubules, in this paper we demonstrate that hydrodynamic shear flow can be used toalign microtubules translocating on a kinesin-coated surface in a direction parallelto the fluid flow. Our evidence supports the hypothesis that the mechanism ofmicrotubule redirection is simply that drag force induced by viscous shear bendsthe leading end of a microtubule, which may be cantilevered beyond its kinesinsupports. This cantilevered end deflects towards the flow direction, until it issubsequently bound to additional kinesins; as translocation continues, the processrepeats until the microtubule is largely aligned with the flow, to a limit determinedby random fluctuations created by thermal energy. We present statistics on therate of microtubule alignment versus various strengths of shear flow as well asconcentrations of kinesin, and also investigate the effects of shear flow on the motility.

Interpreting Correlated Motions Using Normal Mode Analysis

With the increased popularity of normal mode analyses in structural biology, it is important to carefully consider how to best utilize the results for gaining biological insights without over interpretation. The discussion in this article argues that for the purpose of identifying correlated motions in biomolecules, a case separate from concomitant conformational changes of structural motifs, it is generally important to use a large number of normal modes. This is illustrated through three increasingly complex examples. The simplest case includes two bilinearly coupled harmonic oscillators and serves as a straightforward problem where the important considerations are explicit and transparent. The argument is then generalized to include a system of N-coupled harmonic oscillators and finally to a realistic biomolecule. Although a small number of normal modes are useful for probing structural flexibility, it is clear that a much larger number of modes are required for properly investigating correlated motions in biomolecules.

Reversible Switching of Microtubule Motility Using Thermoresponsive Polymer Surfaces

We report a novel approach for the dynamic control of gliding microtubule motility by external stimuli. Our approach is based on the fabrication of a composite surface where functional kinesin motor-molecules are adsorbed onto a silicon substrate between surface-grafted polymer chains of thermoresponsive poly(N-isopropylacrylamide). By external temperature control between 27 and 35 degrees C, we demonstrate the reversible landing, gliding, and releasing of motor-driven microtubules in response to conformational changes of the polymer chains. Our method represents a versatile means to control the activity of biomolecular motors, and other surface-coupled enzyme systems, in bionanotechnological applications.

Modulation of the Viral ATPase Activity by the Portal Protein Correlates with DNA Packaging Efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.

Biomolecular linear motors confined to move upon micro-patterns on glass

Biomolecular linear motor proteins—kinesin and microtubules—are finely patterned on a conventional, flat glass substrate using a Parylene lift-off process. These patterns are useful for single molecule analysis and biohybrid transportation. A patterning width of 2 µm has been achieved with minimal non-specific binding of the proteins. A conventional gliding assay was realized using this glass substrate and behaviors of the single proteins were analyzed.

Toward Devices Powered by Biomolecular Motors

Biomolecular motors can be used in nanometer-scale devices to perform mechanical work. This approach will assist the development of active nanostructures.

Molecular Sorting by Electrical Steering of Microtubules in Kinesin-Coated Channels

Integration of biomolecular motors in nanoengineered structures raises the intriguing possibility of manipulating materials on nanometer scales. We have managed to integrate kinesin motor proteins in closed submicron channels and to realize active electrical control of the direction of individual kinesin-propelled microtubule filaments at Y junctions. Using this technique, we demonstrate molecular sorting of differently labeled microtubules. We attribute the steering of microtubules to electric field-induced bending of the leading tip. From measurements of the orientation-dependent electrophoretic motion of individual, freely suspended microtubules, we estimate the net applied force on the tip to be in the picoNewton range and we infer an effective charge of 12 e- per tubulin dimer under physiological conditions.

Motion control of single F1-ATPase rotary biomolecular motor using microfabricated local heating devices

Biomolecular motors are major targets in single-molecule studies, which reveal molecular behaviors usually hidden in the emsemble- and time-averaging of bulk experiments. Methods for rapid experimental condition control during single-biomolecule observation are a key technology to elucidate the molecular mechanisms of proteins. One of the most promising methods is real-time rapid temperature alternation. A microheater and a microthermosensor were integrated on the glass plate for controlling the temperature locally; the maximum response speeds were 71.5 and 56.9K∕s for temperature rise and fall, respectively. Rapid temperature alternation with microfabricated thermodevice allowed rapid and reversible angular velocity control of a single F1-ATPase, a rotary biomolecular motor. The rapid control of the temperature enabled us to perform rotation assay at temperatures higher than that would “normally” denature them. This revealed that the torque of F1-ATPase seems to increase at higher temperatures with the increasing rate of 4% per 10°C. This method and knowledge for controlling the biomolecular motor can also be applied to future hybrid organic-inorganic nanosystems, which use biomolecular motors as nanoactuators.

Efficient Designs for Powering Microscale Devices with Nanoscale Biomolecular Motors

Current MEMS and microfluidic designs require external power sources and actuators, which principally limit such technology. To overcome these limitations, we have developed a number of microfluidic systems into which we can seamlessly integrate a biomolecular motor, kinesin, that transports microtubules by extracting chemical energy from its aqueous working environment. Here we establish that our microfabricated structures, the self-assembly of the bio-derived transducer, and guided, unidirectional transport of microtubules are ideally suited to create engineered arrays for efficiently powering nano- and microscale devices.

Long-term storage of bionanodevices by freezing and lyophilization

Successful long-term storage of a "smart dust" device integrating biomolecular motors and complex protein assemblies has been demonstrated using freezing or lyophilization, which implies that fabrication and application can be separated even for complex bionanodevices.

Molecular motors, Brownian ratchets, and reflected diffusions

Molecular motors are protein structures that play a central role in accomplishing mechanical work inside a cell. While chemical reactions fuel this work, it is not exactly known how this chemical-mechanical conversion occurs. Recent advances in microbiological techniques have enabled at least indirect observations of molecular motors which in turn have led to significant effort in the mathematical modeling of these motors in the hope of shedding light on the underlying mechanisms involved in intracellular transport. Kinesin which moves along microtubules that are spread throughout the cell is a prime example of the type of motors that are studied in this work. The motion is linked to the presence of a chemical, ATP, but how the ATP is involved in motion is not clearly understood. One commonly used model for the dynamics of kinesin in the biophysics literature is the Brownian ratchet mechanism. In this work, we give a precise mathematical formulation of a Brownian ratchet (or more generally a diffusion ratchet) via an infinite system of stochastic differential equations with reflection. This formulation is seen to arise in the weak limit of a natural discrete space model that is often used to describe motor dynamics in the literature. Expressions for asymptotic velocity and effective diffusivity of a biological motor modeled via a Brownian ratchet are obtained. Linearly progressive biomolecular motors often carry cargos via an elastic linkage. A two-dimensional coupled stochastic dynamical system is introduced to model the dynamics of the motor-cargo pair. By proving that an associated two dimensional Markov process has a unique stationary distribution, it is shown that the asymptotic velocity of a motor pulling a cargo is well defined as a certain Law of Large Number limit, and finally an expression for the asymptotic velocity in terms of the invariant measure of the Markov process is obtained.

Self-assembly driven by molecular motors.

The outcome of a self-assembly process is not only determined by the specified connections between building blocks, but also by the means of bringing building blocks into contact and of testing for the formation of an intended connection. Endowing each building block with the ability to actively move overcomes some limitations of diffusion-driven molecular and nanoscale self-assembly by accelerating transport, reducing unwanted connections, and introducing self-organization phenomena with desirable consequences. Proof-of-principle experiments utilizing biomolecular motors, motor proteins, to propel nanostructures and the underlying concepts are reviewed, and the potential impact for nanomanufacturing is discussed.

Introduction: statistical mechanics of molecular and cellular biological systems

Introduction: statistical mechanics of molecular and cellular biological systems

Biasing the random walk of a molecular motor

Biomolecular motors are often described in mechanical terms, with analogy to cars,turbines, judo throws, levers, etc. It is important to remember however that because oftheir small size, and because of the aqueous environment in which molecular motors move,viscous drag and thermal noise dominate the inertial forces that drive macroscopicmachines. The sequence of motions—conformational changes—by which a motorprotein moves can best be described as a random walk, with transitions fromone state to another occurring by thermal activation over energy barriers. Inthis paper I will address the question of how this random walk is biased by anon-equilibrium chemical reaction (ATP hydrolysis) so that the motor moleculemoves preferentially (with almost unit certainty) in one direction, even when anexternal force is applied to drive it in the opposite direction. I will also discuss howthese ‘soft matter’ motors can achieve thermodynamic efficiencies of nearly 100%.

Packaging actomyosin-based biomolecular motor-driven devices for nanoactuator applications

Biomolecular motors such as the muscle protein myosin with its partner protein actin hold great promise for actuation in hybrid nanoscale biomicroelectromechanical systems devices (bio-MEMS), particularly for future biomedical applications that involve highly localized delivery of biomolecules over short distances (e.g., micrometers) to specific tissue or cellular locations. Two fundamental issues in the construction and packaging of actomyosin-based nanoactuators are the ability to electrically insulate microelectrical components while maintaining both biocompatibility and also compatibility with our functional assays for prototype development and identification of conditions for storage of assembled devices. Here, we show that sputter coating with SiO/sub 2/ provides a straightforward method for electrical insulation that can be readily integrated into existing assays of myosin function in a bio-MEMS setting. We also report using in vitro motility analysis that both rabbit skeletal muscle heavy meromyosin (HMM) and fish (Fundulus heteroclitus) myosin remained functional for at least six days in a bio-MEMS setting when hydrating conditions were maintained. The speed of actin sliding was faster after six days when rabbit HMM was stored in 10% DMSO than in its absence, but there was no effect of DMSO during storage on fish myosin. The speed of actin translocation by fish myosin was significantly increased when adenosine 5'-triphosphate (ATP), the chemical energy source for myosin function, was replaced by the analog 2' deoxy-ATP, as has been previously reported for rabbit HMM. Taken together, these results provide new direction for modulation and control of actomyosin-based nanoactuators and also for long-term storage of assembled nanoactuators.

Caged ATP-fuel for bionanodevices

Micro- and nanodevices require the controlled delivery of energy to power a variety of processes. The current paradigm of connecting a miniaturized device to a set of macroscopic auxiliary devices, such as power supplies or pumps, for the delivery of electrical and mechanical energy needs to be replaced to enable the design of stand-alone integrated bionanodevices with applications in remote biosensing or nanomedicine. Biological nanomachines, such as the motor protein kinesin, can efficiently convert energy stored in chemical compounds, in particular adenosine 5'-triphosphate (ATP), into mechanical work. This ability is an attractive feature of hybrid devices powered by biomolecular motors, since it removes the need for the storage and conversion of electrical energy. The consequences are a simplified fabrication process and packaging, leading to higher yields and lower costs, and the broadening of the applications, which can now include field-deployable nanodevices. Here, the potential of caged ATP as fuel for such engineering applications is discussed. Caged ATP can be stored in the buffer solution of a bionanodevice, "uncaged" by UV light, and utilized as fuel by many enzymes to catalyze chemical changes or power active transport. We demonstrate that DMNPE-caged ATP can be stored in sufficient amounts in a typical device and that the activation can be triggered with a UV lamp or even sunlight.

Microfabricated capped channels for biomolecular motor-based transport

Kinesins are molecular motors that transport intracellular cargo along microtubules and provide a model system for force generation that can be exploited for biomotor powered nano- and micro-machines. To use this biological system for microscale transport, the most common approach is to reverse the biological geometry and move microtubules along surfaces functionalized with kinesin motors. The microtubules then become potential transport vehicles for sensors and lab-on-a-chip applications. A key requirement for extracting useful work from this system is confinement and control of microtubule movements over kinesin-coated surfaces. The open channel approaches used to date are limited because microtubules that lose contact with the kinesin motors rapidly diffuse away. As a step toward making stand-alone devices incorporating kinesin motors and microtubules, we have developed methods to fabricate capped channels that provide three-dimensional microtubule confinement. We first tested the activity of kinesin motors on a range of surfaces and found that motors were functional on a number of hydrophilic surfaces and nonfunctional on hydrophobic surfaces. In this work, SU-8 photoresist is used to fabricate open channels and a layer of bisbenzocyclobutene (BCB) or dry-film photoresist is used to encapsulate the channels. To allow sample introduction, we fabricate a hierarchical series of microfluidic channels. In this approach, macroscale (/spl sim/250-/spl mu/m) channels in glass or silicon substrates are used to hold fine-gauge stainless steel tubing and allow connection to various fluid sources and intermediate scale (/spl sim/50-/spl mu/m) channels fabricated in thick (/spl sim/50-/spl mu/m) dry-film photoresist are used to connect the macroscale channels to microscale (1-15-/spl mu/m) SU-8 photoresist channels. This paper is the first demonstration of kinesin-based microtubule transport in enclosed microfluidic channels and provides an important step toward packaging these biomolecular motors into functional devices.