Power Stroke Mechanism Research Articles

Four phases of a force transient emerge from a binary mechanical system.

Accurate models of muscle contraction are important for understanding both muscle performance and the therapeutics that enhance physiological function. However, models are only accurate and meaningful if they are consistent with physical laws. A single muscle fiber contains billions of randomly fluctuating atoms that on the spatial scale of a muscle fiber generate unidirectional force and power output. This thermal system is formally constrained by the laws of thermodynamics, and a recently developed thermodynamic model of muscle force generation provides qualitative descriptions of the muscle force-velocity relationship, muscle force generation, muscle force transients, and the thermodynamic work loop of muscle with a thermodynamic (not molecular) power stroke mechanism. To demonstrate the accuracy of this model requires that its outputs be quantitatively compared with experimentally observed muscle function. Here I show that a two-state thermodynamic model accurately describes the experimentally observed four-phase force transient response to both mechanical and chemical perturbations. This is the simplest possible model of one of the most complex characteristic signatures of muscle mechanics.

Kinetic Asymmetry and Directionality of Nonequilibrium Molecular Systems.

Scientists have long been fascinated by the biomolecular machines in living systems that process energy and information to sustain life. The first synthetic molecular rotor capable of performing repeated 360° rotations due to a combination of photo- and thermally activated processes was reported in 1999. The progress in designing different molecular machines in the intervening years has been remarkable, with several outstanding examples appearing in the last few years. Despite the synthetic accomplishments, there remains confusion regarding the fundamental design principles by which the motions of molecules can be controlled, with significant intellectual tension between mechanical and chemical ways of thinking about and describing molecular machines. A thermodynamically consistent analysis of the kinetics of several molecular rotors and pumps shows that while light driven rotors operate by a power-stroke mechanism, kinetic asymmetry-the relative heights of energy barriers-is the sole determinant of the directionality of catalysis driven machines. Power-strokes-the relative depths of energy wells-play no role whatsoever in determining the sign of the directionality. These results, elaborated using trajectory thermodynamics and the nonequilibrium pump equality, show that kinetic asymmetry governs the response of many non-equilibrium chemical phenomena.

Theoretical Estimates of Physical Aspects Involved in the Power Stroke Hypothesis for Ribosome Translocation

The power stroke mechanism for ribosome translocation is evaluated through calculations of basic aspects of the mechanics, hydrodynamics, and elasticity involved in such a process. The results show that reported power stroke magnitudes would generate several physical problems for ribosome translocation: unwanted or disproportionate displacements of the ribosome and mRNA, destabilizing competing forces and high tension on the mRNA in the ribosome tunnel. All these issues can be related to the ribosome losing its correct reading frame during mRNA translation. To improve ribosome translocation models, the suggestion is to change the focus from tRNA–mRNA forced displacement to ribosome motion along the mRNA. Also, to consider that the mRNA is essentially different from cytoskeleton fibers and that the ribosome does not work alone during mRNA translation.

Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease

Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.

Using Catalysis to Drive Chemistry Away from Equilibrium: Relating Kinetic Asymmetry, Power Strokes, and the Curtin-Hammett Principle in Brownian Ratchets.

Chemically fueled autonomous molecular machines are catalysis-driven systems governed by Brownian information ratchet mechanisms. One fundamental principle behind their operation is kinetic asymmetry, which quantifies the directionality of molecular motors. However, it is difficult for synthetic chemists to apply this concept to molecular design because kinetic asymmetry is usually introduced in abstract mathematical terms involving experimentally inaccessible parameters. Furthermore, two seemingly contradictory mechanisms have been proposed for chemically driven autonomous molecular machines: Brownian ratchet and power stroke mechanisms. This Perspective addresses both these issues, providing accessible and experimentally useful design principles for catalysis-driven molecular machinery. We relate kinetic asymmetry to the Curtin-Hammett principle using a synthetic rotary motor and a kinesin walker as illustrative examples. Our approach describes these molecular motors in terms of the Brownian ratchet mechanism but pinpoints both chemical gating and power strokes as tunable design elements that can affect kinetic asymmetry. We explain why this approach to kinetic asymmetry is consistent with previous ones and outline conditions where power strokes can be useful design elements. Finally, we discuss the role of information, a concept used with different meanings in the literature. We hope that this Perspective will be accessible to a broad range of chemists, clarifying the parameters that can be usefully controlled in the design and synthesis of molecular machines and related systems. It may also aid a more comprehensive and interdisciplinary understanding of biomolecular machinery.

Effect of Binding-Affinity and ATPase Activity on the Velocities of Kinesins Using Ratchet Models.

We use two-state ratchet models containing single and coupled Brownian motors to understand the role of motor-microtubule binding, ATPase reaction rate and dimerisation on the translational velocities of Kinesin motors. We use model parameters derived from the experimental measurements on KIF1A, KIF13A, KIF13B, and KIF16B motors to compute velocities in μm/s. We observe that both the models show the same trend in velocities (KIF1A > KIF13A > KIF13B > KIF16B) as the experimental results. However, the models significantly underpredict the velocities when compared with the experiments. The predictions of the coupled-motor model are closer to the experiments than those of the single-motor model. Our results indicate that the variation of ATPase reaction rate governs the trend in velocities for the above four motors. The variation of motor-microtubule binding affinity and the coupling strength between the motor domains may only have a secondary effect. More rigorous models that incorporate the power-stroke mechanism are necessary for better quantitative compliance with the experiments.

Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP.

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG–SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

Effect of myofibril passive elastic properties on the mechanical communication between motor proteins on adjacent sarcomeres

Rapid sarcomere lengthening waves propagate along a single muscle myofibril during spontaneous oscillatory contraction (SPOC). In asynchronous insect flight muscles, SPOC is thought to be almost completely synchronized over the entire myofibril. This phenomenon does not require Ca2+ regulation of the dynamics of the motor proteins, and cannot be explained simply by the longitudinal mechanical equilibrium among sarcomeres in the myofibril. In the present study, we rationalize these phenomena by considering the lateral mechanical equilibrium, in which two tensions originating from the inverse relationship between sarcomere length and lattice spacing, along with the lattice alignment, play important roles in the mechanical communication between motor proteins on adjacent filaments via the Z-disc. The proposed model is capable of explaining various SPOC phenomena based on the stochastic power-stroke mechanism of motor proteins, which responds to temporal changes in longitudinal mechanical load.

Protein translocation by the SecA ATPase occurs by a power-stroke mechanism.

SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides.

Atomically Detailed Simulation of the Powerstroke in Myosin II by Milestoning

The interaction between actin and myosin II plays an important role in a variety of cellular functions. In particular, myosin II is involved in muscle contraction, which is attributed to the sliding of thin filament actin past the thick myosin II filaments. Past studies on the structure of myosin have linked severe pathologies to defects in myosin, making it important to understand the mechanism of the system. In this study, we focus our analysis on the powerstroke of the myosin II cross bridge cycle, which is the active process of muscle contraction. To do this, we use an algorithm called Milestoning which partitions the dynamics into a sequence of trajectories between “milestones” along the reaction pathway. The structure of myosin II bound to actin in the rigor state was used as a starting point, and a structure for the bound pre-powerstroke state was developed using existing published structures for the unbound pre-powerstroke state as well as experimental data gathered about the movement of myosin II during the powerstroke. With both the beginning and final states of the powerstroke, we can interpolate between these structures to build intermediate states along the reaction pathway. A total of 97 all-atom structures along the pathway of the powerstroke were developed to serve as guides and “anchors” along the reaction pathway. Milestoning short trajectories between cells defined by the anchors will allow for the computation thermodynamics and kinetics of the myosin II powerstroke. This work will lead to a significant improvement in our understanding of the complete powerstroke mechanism, which will in turn facilitate future research on the effects of structural defects in myosin II on powerstroke function and muscle contraction.

Elastic coupling power stroke mechanism of the F1-ATPase molecular motor

The angular velocity profile of the 120° F1-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a ΔμATP = -31.25 kBT at a time resolution of 10 μs. Angular velocities during the first 60° of the power stroke (phase 1) varied inversely with temperature, resulting in negative activation energies with a parabolic dependence. This is direct evidence that phase 1 rotation derives from elastic energy (spring constant, κ = 50 kBT·rad-2). Phase 2 of the power stroke had an enthalpic component indicating that additional energy input occurred to enable the γ-subunit to overcome energy stored by the spring after rotating beyond its 34° equilibrium position. The correlation between the probability distribution of ATP binding to the empty catalytic site and the negative Ea values of the power stroke during phase 1 suggests that this additional energy is derived from the binding of ATP to the empty catalytic site. A second torsion spring (κ = 150 kBT·rad-2; equilibrium position, 90°) was also evident that mitigated the enthalpic cost of phase 2 rotation. The maximum ΔGǂ was 22.6 kBT, and maximum efficiency was 72%. An elastic coupling mechanism is proposed that uses the coiled-coil domain of the γ-subunit rotor as a torsion spring during phase 1, and then as a crankshaft driven by ATP-binding-dependent conformational changes during phase 2 to drive the power stroke.

Overview of the mechanism of cytoskeletal motors based on structure.

In the last two decades, a wealth of structural and functional knowledge has been obtained for the three major cytoskeletal motor proteins, myosin, kinesin and dynein, which we review here. The cytoskeletal motor proteins myosin and kinesin are structurally similar in the core architecture of their motor domains and have similar force-producing mechanisms that are coupled with the chemical cycles of ATP binding, hydrolysis, Pi release and subsequent ADP release. The force is generated through conformational changes in the motor domain during Pi release and ATP binding in myosin and kinesin, respectively, and then converted into the rotation of the lever arm or neck linker (referred to as a power stroke) through the common structural pathways. On the other hand, the dynein cytoskeletal motor is an AAA+ protein and has a different structure and power stroke mechanism from those of myosins and kinesins. The linker protruding from the AAA+ ring of dynein swings according to the ATPase states, which, presumably, generates force to carry cargos within a cell. The communication mechanism between the track-binding and ATPase domains of dynein is unique because the two helices that presumably slide with respect to each other work as coordinators for these domains. Details of the mechanism underlying the power stroke and interdomain communication were revealed through recent progress in the structural studies of myosin, kinesin and dynein.

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.

Simulating the dynamics of the mechanochemical cycle of myosin-V

The detailed dynamics of the cycle of myosin-V are explored by simulation approaches, examining the nature of the energy-driven motion. Our study started with Langevin dynamics (LD) simulations on a very coarse landscape with a single rate-limiting barrier and reproduced the stall force and the hand-over-hand dynamics. We then considered a more realistic landscape and used time-dependent Monte Carlo (MC) simulations that allowed trajectories long enough to reproduce the force/velocity characteristic sigmoidal correlation, while also reproducing the hand-over-hand motion. Overall, our study indicated that the notion of a downhill lever-up to lever-down process (popularly known as the powerstroke mechanism) is the result of the energetics of the complete myosin-V cycle and is not the source of directional motion or force generation on its own. The present work further emphasizes the need to use well-defined energy landscapes in studying molecular motors in general and myosin in particular.

Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.

Walk and Check along a Viral RNA Polymerase Transcription Elongation Path

Gene transcription is a fundamental step in the central dogma of molecular biology. The essential protein enzymes that direct the process are RNA polymerases (RNAPs). The viral RNAP from bacteriophage T7 serves a self-sufficient transcription engine and is widely used for lab gene expression and engineering, although its operational mechanisms remain controversial. We have systematically studied mechano-chemical coupling and fidelity control mechanisms of T7 RNAP during its elongation. Based on single molecule measurements and high-resolution structures, we had demonstrated that a small translocation free energy bias aids nucleotide selection in T7 RNAP [1]. Accordingly, we performed atomistic molecular dynamics (MD) simulation and discovered how a critical tyrosine residue aids the nucleotide selection for selective Brownian ratcheting of the RNAP [2]. In addition, we also built a theoretical framework to analyze efficient stepwise nucleotide selection of the RNAP in the absence of proofreading [3]. Recently, we constructed the Markov state model for the PPi product release and translocation of T7 RNAP implementing extensive MD simulations. In particular, we noticed a universal module in which an essential lysine/arginine residue greatly assists a jump-from-cavity activation process of the PPi release [4]. The activated PPi release is unlikely to drive the RNAP translocation, thus ruling out a power stroke mechanism during the RNAP elongation. Overall, our work demonstrates how physical modeling and large-scale simulations can be combined to reveal underlying mechanisms of a key physiological process that may not be easily accessible from experimental approaches.[1] J Yu, G Oster. Biophys J, 102, 532, 2012.[2] B Duan et al., J Yu. Biophys J, 107, 2130, 2014.[3] J Yu. Mol Based Math Biol, 2,141, 2014.[4] LT Da et al., J Yu. ms under review 2015.

Resolving Conformational Switches in the Protease Ftsh using Single-Molecule FRET

FtsH is a homohexameric AAA+ protease embedded in the prokaryote membrane, where it recognizes, unfolds, translocates and lyses protein substrates. Previous crystal structural data of Thermotoga maritima FtsH in an ADP-bound and an apo-state showed an ATPase and a protease domain linked by a flexible unresolved hinge, facilitating a large conformational change upon ADP binding. The current model of the chemo-mechanical cycle was inferred from these two crystal structures and describes FtsH with a power stroke mechanism, whereby the energy released by ATP hydrolysis is converted into a conformational switch. To date, this mechanism could not be observed experimentally. Here, we use single-molecule Förster Resonance Energy Transfer (smFRET) to study FtsH in real time. Our assay involved the vesicle encapsulation of unlabeled and double-labeled monomers to promote self-assembled FtsH hexamers. Total Internal Reflection Fluorescence (TIRF) microscopy of the immobilized molecules allowed us to collect kinetic information of conformational changes. Data analysis based on hidden Markov modeling resolved distinct conformational states, transition coordinates and transition rates. Surprisingly, without nucleotide, an FtsH monomer adopted four distinct conformational states, undergoing highly dynamic interconversion on the second timescale. Thus, FtsH performed stochastic transitions independently of an energy source, but rather using thermal activation. Upon binding of ATP, the number of conformational states remained identical, however, presence of ATP reduced some barriers of the free energy landscape. A homolog mutation in FtsH, A359V, of the pathogenic mutation A510V in paraplegin displayed an additional further closed conformational state, which was highly populated in the presence of ATP. We propose that this highly populated state could reflect the malfunctioning of the pathogenic mutation in paraplegin.

PPi Release Followed by DNA Translocation Studied from Atomistic Simulations of T7 RNA Polymerase Transcription

Bacteriophage T7 RNA polymerase (RNAP) serves a prototypical system to study general physical mechanisms of transcription due to its relative simple structures along with self-sufficient and strong transcription activities. We computationally study the pyrophosphate ion (PPi) product release and DNA translocation processes during the T7 RNAP elongation with unprecedented structural dynamics detail. We implemented extensive all-atom molecular dynamics (MD) simulations and constructed the Markov state model (MSM). The MSM reveals a jump-from-cavity PPi release mechanism, which distinguishes from charge facilitated hopping mechanisms identified previously for bacterial and eukaryotic multi-subunit RNAPs. Furthermore, we performed steered MD simulations and microsecond long MD simulations to explore additional couplings and slow motions. We found that the PPi release does not appear to be tightly coupled to opening of an O-helix that is directly tied to the DNA translocation. Hence, the study disfavors a power stroke mechanism of the RNAP elongation. In particular, we discovered a key lysine/arginine residue that assists the PPi release and the module appears to be universal not only to structurally similar polymerases but also to the multi-subunit RNAPs, even though their overall structures and mechanisms look quite different. We also want to understand why the viral RNAP does not backtrack during its Brownian ratcheting.

Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines.

Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.