Single Molecular Motor Research Articles

Effect of load-resisting force on photoisomerization mechanism of a single second generation light-driven molecular rotary motor.

A pivotal aspect of molecular motors is their capability to generate load capacity from a single entity. However, few studies have directly characterized the load-resisting force of a single light-driven molecular motor. This research provides a simulation analysis of the load-resisting force for a highly efficient, second-generation molecular motor developed by Feringa et al. We investigate the M-to-P photoinduced nonadiabatic molecular dynamics of 9-(2,3-dihydro-2-methyl-1H-benz[e]inden-1-ylidene)-9H-fluorene utilizing Tully's surface hopping method at the semi-empirical OM2/MRCI level under varying load-resisting forces. The findings indicate that the quantum yield remains relatively stable under forces up to 0.003a.u., with the photoisomerization mechanism functioning typically. Beyond this threshold, the quantum yield declines, and an alternative photoisomerization mechanism emerges, characterized by an inversion of the central double bond's twisting direction. The photoisomerization process stalls when the force attains a critical value of 0.012a.u. Moreover, the average lifetime of the excited state oscillates around that of the unperturbed system. The quantum yield and mean lifetime of the S1 excited state in the absence of external force are recorded at 0.54 and 877.9fs, respectively. In addition, we analyze a time-dependent fluorescence radiation spectrum, confirming the presence of a dark state and significant vibrations, as previously observed experimentally by Conyard et al.

Bending Actuation of Hydrogels through Rotation of Light‐Driven Molecular Motors

AbstractThe unidirectional rotation of chemically crosslinked light‐driven molecular motors is shown to progressively shift the swelling equilibrium of hydrogels. The concentration of molecular motors and the initial strand density of the polymer network are key parameters to modulate the macroscopic contraction of the material, and both parameters can be tuned using polymer chains of different molecular weights. These findings led to the design of optimized hydrogels revealing a half‐time contraction of approximately 5 min. Furthermore, under inhomogeneous stimulation, the local contraction event was exploited to design useful bending actuators with an energy output 400 times higher than for previously reported self‐assembled systems involving rotary motors. In the present configuration, we measure that a single molecular motor can lift up loads of 200 times its own molecular weight.

Bending Actuation of Hydrogels through Rotation of Light-Driven Molecular Motors.

The unidirectional rotation of chemically crosslinked light-driven molecular motors is shown to progressively shift the swelling equilibrium of hydrogels. The concentration of molecular motors and the initial strand density of the polymer network are key parameters to modulate the macroscopic contraction of the material, and both parameters can be tuned using polymer chains of different molecular weights. These findings led to the design of optimized hydrogels revealing a half-time contraction of approximately 5 min. Furthermore, under inhomogeneous stimulation, the local contraction event was exploited to design useful bending actuators with an energy output 400 times higher than for previously reported self-assembled systems involving rotary motors. In the present configuration, we measure that a single molecular motor can lift up loads of 200 times its own molecular weight.

Fluid flow in the sarcomere

A highly organized and densely packed lattice of molecular machinery within the sarcomeres of muscle cells powers contraction. Although many of the proteins that drive contraction have been studied extensively, the mechanical impact of fluid shearing within the lattice of molecular machinery has received minimal attention. It was recently proposed that fluid flow augments substrate transport in the sarcomere, however, this analysis used analytical models of fluid flow in the molecular machinery that could not capture its full complexity. By building a finite element model of the sarcomere, we estimate the explicit flow field, and contrast it with analytical models. Our results demonstrate that viscous drag forces on sliding filaments are surprisingly small in contrast to the forces generated by single myosin molecular motors. This model also indicates that the energetic cost of fluid flow through viscous shearing with lattice proteins is likely minimal. The model also highlights a steep velocity gradient between sliding filaments and demonstrates that the maximal radial fluid velocity occurs near the tips of the filaments. To our knowledge, this is the first computational analysis of fluid flow within the highly structured sarcomere.

The Measurement of the Stepping Force of Purified Zymogen Graunles using a Single Beam Optical Trap

The transportation of vesicles is important for the development, organization, and functioning of all cellular organisms. The failures of vesicle transportation cause many diseases and chronic illnesses associated with the malfunctioning of these systems. The molecular motors responsible for these vesicle transportations supply the energy for locomotion by exchanging chemical energy into mechanical force. A particular vesicle, zymogen granules (ZGs), digestive enzymes secreted by the pancreas are studied. Recently, the mass spectroscopy experiment showed that myosin Ib, myosin VIIb, and Myosin Vc were found on the ZGs. To understand what the molecular motors involved in the vesicle trafficking, we purify ZGs from rat pancreas and examine the stepping manner and force that is used by a single beam optical tweezer. The average speed of the ZGs is around 100 nm/sec, which is similar to our previous study of two myosin Vc molecules tagged with DNA. The results indicate that the stepping is unidirectional with a 16-nm to 72-nm step-size, while some of the steps are a back-and-force ensemble stepping along the axis of actin filaments. The average force and maximum force in the optical trap are 0.1 pN and 10 pN, respectively. The typical force of a single molecular motor has a range of 1 pN to 5 pN. Thus, our finding in the unidirectional movement can be created by a single molecule motor. Our results suggest that most steps maybe created by myosin Vc.

Processivity of molecular motors under vectorial loads.

Molecular motors are cellular machines that drive the spatial organization of the cells by transporting cargos along intracellular filaments. Although the mechanical properties of single molecular motors are relatively well characterized, it remains elusive how the geometry of a load imposed on a motor affects its processivity, i.e., the average distance that a motor moves per interaction with a filament. Here, we theoretically explore this question for a single-kinesin molecular motor by analyzing the load dependence of the stepping and detachment processes. We find that the processivity of the kinesin increases with lowering the load angle between the kinesin and the microtubule filament, due to the deceleration of the detachment rate. When the load angle is large, the processivity is predicted to enhance with accelerating the stepping rate through an optimal distribution of the load over the kinetic transition rates underlying a mechanical step of the motor. These results provide new insights into understanding of the design of potential synthetic biomolecular machines that can travel long distances with high velocities.

Bacterial flagellar motor as a multimodal biosensor.

Bacterial Flagellar Motor is one of nature's rare rotary molecular machines. It enables bacterial swimming and it is the key part of the bacterial chemotactic network, one of the best studied chemical signalling networks in biology, which enables bacteria to direct its movement in accordance with the chemical environment. The network can sense down to nanomolar concentrations of specific chemicals on the time scale of seconds. Motor's rotational speed is linearly proportional to the electrochemical gradients of either proton or sodium driving ions, while its direction is regulated by the chemotactic network. Recently, it has been discovered that motor is also a mechanosensor. Given these properties, we discuss the motor's potential to serve as a multifunctional biosensor and a tool for characterising and studying the external environment, the bacterial physiology itself and single molecular motor biophysics.

The Direct Measurement of the Stepping Force of a Purified Vesicle using a Single Beam Optical Trap

The transportation of vesicles is important for the development, organization, and functioning of all cellular organisms. The failure of vesicle transportation can cause many diseases and chronic illnesses associated with the malfunctioning of these systems. The molecular motors responsible for this vesicle transportation supply the energy for locomotion by exchanging chemical energy into mechanical force. Particular vesicles, zymogen granules (ZGs), are digestive enzymes secreted by the pancreas. Recently, mass spectroscopy experiments have shown that Myosin Ib, Myosin VIIb, and Myosin Vc are found on ZGs. To understand the molecular motors involved in the vesicle trafficking, we observed the in vitro motility of purified ZGs from rat pancreas and examine the stepping manner and force that is produced using a single beam optical trap. The average speed of the ZGs is around 100 nm/sec, which is similar to our previous study of two myosin Vc molecules tagged with DNA. The stepping seems to have patterns; one is unidirectional steps with 16-nm to 72-nm step-size, while some steps have a back-and-forth ensemble stepping along the axis of actin filaments. The average force and maximum force in the optical trap are 0.3 ± 0.1 pN and 0.5 ± 0.2 pN, respectively. The typical force of a single molecular motor has a range of 1 pN to 5pN. Thus, our finding in the unidirectional movement can be created by a single motor. Our results conclud that most steps may be created by myosin Vc.

Limited processivity of single motors improves overall transport flux of self-assembled motor-cargo complexes.

Single kinesin molecular motors can processively move along a microtubule (MT) a few micrometers on average before dissociating. However, cellular length scales over which transport occurs are several hundred microns and more. Why seemingly unreliable motors are used to transport cellular cargo remains poorly understood. We propose a theory for how low processivity, the average length of a single bout of directed motion, can enhance cellular transport when motors and cargos must first diffusively self-assemble into complexes. We employ stochastic modeling to determine the effect of processivity on overall cargo transport flux. We show that, under a wide range of physiologically relevant conditions, possessing "infinite" processivity does not maximize flux along MTs. Rather, we find that lowering processivity, i.e., weaker binding of motors to MTs, can improve transport flux. These results shed light on the relationship between processivity and transport efficiency and offer a theory for the physiological benefits of low motor processivity.

Phosphate increase during fatigue affects crossbridge kinetics in intact mouse muscle at physiological temperature.

Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi ] in the myoplasm. High [Pi ] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi ] was raised by fatiguing the fibres with a series of tetanic contractions. [Pi ] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low- or no-force state and show that fibre length changes perturb the Pi -sensitive force generation of the crossbridge cycle. Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [Pi ] in the myoplasm. Experiments in skinned fibres at low temperature (10-12°C) have shown that [Pi ] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of Pi decrease with temperature and this raises the question of the role of Pi under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [Pi ] without significant Ca2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [Pi ] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics.

Distinctions between dynamic characteristics of the single EG5 motor protein along neural vs. cancerous microtubules

The kinesin 5 motor contributes critically to mitosis, and is often upregulated in cancer. In vitro motility studies of kinesin 5 moving along bovine brain microtubules indicate that the motors have limited processivity. Cancer cells have abnormal mitotic behavior, so one might wonder whether the functional properties of kinesin 5 change in such a background. Because there could be multiple unknown changes in cancerous vs normal cells, we chose to address this question in a controlled in vitro environment. Specifically, through a series of parallel experiments along bovine brain vs. breast cancer microtubules, we quantified the in vitro motility characteristics of single Eg5 molecular motors along these two types of microtubules, combining the utilization of an optical trapping technique with a study of motion in the unloaded regime. The obtained values indicate that Eg5 processivity is 40% less along MCF7 microtubules, compared to that measured on bovine brain MTs. Interestingly, not all single-molecule properties are altered, as the velocity of the single motor doesn't show any significant changes on either track, though the binding time along MCF7 microtubules is almost 25% shorter. The current results, in conjunction with our previously reported outcomes of the evaluation of the Eg5's characteristics under external load, show that in transition from no-load to high-load regime, the Eg5 binding time has less sensitivity on MCF7 as compared to bovine brain MTs. This finding is intriguing, as it suggests that, potentially, groups of Eg5 motors function more effectively in the cancer background of a large ensemble, possibly contributing to faster mitosis in cancer cells.

Single-Molecule Total Internal Reflection Fluorescence Microscopy.

The advent of total internal reflection fluorescence (TIRF) microscopy has permitted visualization of biological events on an unprecedented scale: the single-molecule level. Using TIRF, it is now possible to view complex biological interactions such as cargo transport by a single molecular motor or DNA replication in real time. TIRF allows for visualization of single molecules by eliminating out-of-focus fluorescence and enhancing the signal-to-noise ratio. TIRF has been instrumental for studying in vitro interactions and has also been successfully implemented in live-cell imaging. Visualization of cytoskeletal structures and dynamics at the plasma membrane, such as endocytosis, exocytosis, and adhesion, has become much clearer using TIRF microscopy. Thanks to recent advances in optics and commercial availability, TIRF microscopy is becoming an increasingly popular and user-friendly technique. In this introduction, we describe the fundamental properties of TIRF microscopy and the advantages of using TIRF for single-molecule investigation.

A Simple Experimental Model to Investigate Force Range for Membrane Nanotube Formation

The presence of membrane tubules in living cells is essential to many biological processes. In cells, one mechanism to form nanosized lipid tubules is via molecular motor induced bilayer extraction. In this paper, we describe a simple experimental model to investigate the forces required for lipid tube formation using kinesin motors anchored to DOPC vesicles. Previous related studies have used molecular motors actively pulling on the membrane to extract a nanotube. Here, we invert the system geometry; molecular motors are used as static anchors linking DOPC vesicles to a two-dimensional microtubule network and an external flow is introduced to generate nanotubes facilitated by the drag force. We found that a drag force of ~7 pN was sufficient for tubule extraction for vesicles ranging from 1 to 2 um in radius. By our method, we found that the force generated by a single molecular motor was sufficient for membrane tubule extraction from a spherical lipid vesicle.

Light-Driven Surface Patterning of Supramolecular Polymers with Extremely Low Concentration of Photoactive Molecules.

Light-induced surface patterning in azobenzene-containing polymers and other materials is a widely studied phenomenon with possible applications in fields ranging from photonics to biology. Yet, the fundamental understanding of this purely photodriven mass transport remains inadequate, and existing literature fails to define a threshold chromophore content for mass transport to occur, if such a limit exists. This letter presents a systematic study of the relationship between chromophore concentration and mass transport using hydrogen-bonded polymer-azobenzene complexes, in which the chromophore concentration can be freely adjusted while keeping the polymer backbone unchanged. Essentially, we demonstrate that effective surface patterning can be induced even at an extremely low chromophore content of 1 mol %, when only every tenth polymer chain carries a single azobenzene molecular motor. Importantly, the results highlight the extraordinary photomechanical power of azobenzene and contribute to the fundamental understanding of the light-induced motions.

Ultrafast Force-Clamp Spectroscopy of Single Molecular Motors and DNA Binding Proteins

Force plays a fundamental role in a wide array of biological processes, regulating, for example, enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. Here, we present a novel force-clamp laser trap that is capable of applying constant loads to molecular complexes with lifetimes above ∼10 μs [Capitanio et al., Nature Methods 9, 1013-1019 (2012)]. Such capability is enhanced by a detection strategy allowing the investigation of very short interactions (∼100μs). Moreover, the high temporal and spatial resolution of the method enables us to probe sub-nanometer conformational changes with a time resolution of few tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. These results resolve the molecular mechanism underlying regulation of muscle contraction by force. Our technique could be straightforwardly applied to a wide variety of non-processive molecular motors, single domains of processive motors, protein-DNA and protein-RNA interactions, and conceivably to any short-lived protein-protein interaction, opening new avenues for investigating the effects of forces on biological processes.

A Little Engine That Could: ATP-Powered Electrical Battery and Heater Inside Cells

A Little Engine That Could: ATP-Powered Electrical Battery and Heater Inside Cells

Reconstructing the free-energy landscape associated to molecular motors processivity

We propose a biochemical model providing the kinetic and energetic descriptions of the processivity dynamics of kinesin and dinein molecular motors. Our approach is a modified version of a well known model describing kinesin dynamics and considers the presence of a competitive inhibition reaction by ADP. We first reconstruct a continuous free-energy landscape of the cycle catalyst process that allows us to calculate the number of steps given by a single molecular motor. Then, we calculate an analytical expression associated to the translational velocity and the stopping time of the molecular motor in terms of time and ATP concentration. An energetic interpretation of motor processivity is discussed in quantitative form by using experimental data. We also predict a time duration of collective processes that agrees with experimental reports.

Stochastic thermodynamics of single enzymes and molecular motors

For a single enzyme or molecular motor operating in an aqueous solution of non-equilibrated solute concentrations, a thermodynamic description is developed on the level of an individual trajectory of transitions between states. The concept of internal energy, intrinsic entropy and free energy for states follows from a microscopic description using one assumption on time scale separation. A first-law energy balance then allows the unique identification of the heat dissipated in one transition. Consistency with the second law on the ensemble level enforces both stochastic entropy as third contribution to the entropy change involved in one transition and the local detailed balance condition for the ratio between forward and backward rates for any transition. These results follow without assuming weak coupling between the enzyme and the solutes, ideal solution behavior or mass action law kinetics. The present approach highlights both the crucial role of the intrinsic entropy of each state and the physically questionable role of chemiostats for deriving the first law for molecular motors subject to an external force under realistic conditions.

Improved Hidden Markov Models for Molecular Motors, Part 2: Extensions and Application to Experimental Data

Unbiased interpretation of noisy single molecular motor recordings remains a challenging task. To address this issue, we have developed robust algorithms based on hidden Markov models (HMMs) of motor proteins. The basic algorithm, called variable-stepsize HMM (VS-HMM), was introduced in the previous article. It improves on currently available Markov-model based techniques by allowing for arbitrary distributions of step sizes, and shows excellent convergence properties for the characterization of staircase motor timecourses in the presence of large measurement noise. In this article, we extend the VS-HMM framework for better performance with experimental data. The extended algorithm, variable-stepsize integrating-detector HMM (VSI-HMM) better models the data-acquisition process, and accounts for random baseline drifts. Further, as an extension, maximum a posteriori estimation is provided. When used as a blind step detector, the VSI-HMM outperforms conventional step detectors. The fidelity of the VSI-HMM is tested with simulations and is applied to in vitro myosin V data where a small 10 nm population of steps is identified. It is also applied to an in vivo recording of melanosome motion, where strong evidence is found for repeated, bidirectional steps smaller than 8 nm in size, implying that multiple motors simultaneously carry the cargo.

Synthesis of Extended Nanoscale Optical Encoders

An optical encoder is a device that uses an interrupted light source-sensor pair to map linear or rotational motion onto a periodic signal. Simple, inexpensive optical encoders are used for precise positioning in machines such as desktop printers, disk drives, and astronomical telescopes. A strand of DNA labeled with a series of Förster resonance energy transfer acceptor dyes can perform the same function at the nanometer scale, producing a periodic fluorescence signal that encodes the movement of a single donor-labeled molecular motor with high spatial and temporal resolution. Previous measurements of this type have employed encoders limited to five acceptor dyes, and hence five signal periods, restricting the range of motion that could be followed. Here we describe two methods for synthesizing double-stranded DNA containing several to hundreds of regularly spaced dyes on one strand. Distinct functional groups incorporated at the encoder ends enable tethering for single-molecule measurements.