Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy

Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post-to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its postto prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ∼2-3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors.yosin constitutes a diverse class of molecular motors that perform mechanical work by producing movement on the actin filaments. These cellular machines tightly couple various steps of the ATP hydrolysis and product release cycle with mechanical force generation on actin and can therefore perform coordinated movements within the cell (1-3). Classical examples of myosin motors constitute the muscle protein that performs a single mechanical force generation cycle and then detaches from the actin filament. These molecules work in large numbers to produce a net directed movement of the actin during muscle contraction (2). Furthermore, living cells have used the basic functional cycle of myosin to generate several other members of the myosin family, some of which produce repetitive mechanochemical movements on the actin filaments. The most studied among these processive motors is myosin V, which is responsible for carrying cargo to different destinations using the intracellular network of actin filaments (4). At the heart of all types of myosin action is its ability to couple large conformational changes with ATP hydrolysis and actin binding/release cycles to eventually perform a unidirectional motion (3). Although some features of the cycle are well understood, one of the most intriguing remaining problems is to unravel the factors that allow myosin V to walk in only one direction toward...

Section: Iii3 Modeling the Energetics Behind The Unidirectional Myomentioning

confidence: 99%

Electrostatic origin of the unidirectionality of walking myosin V motors

Mukherjee

Warshel

2013

“…Moreover, polarization-resolved single-molecule experiments have previously been used to study the orientations of chromophores embedded in complex systems, including glasses (16,17), biological macromolecules (18)(19)(20), conjugated polymers (21), and light-harvesting complexes (22). The majority of these methods are capable of sampling a single-molecule signal with time resolution on the order of tens of milliseconds or longer.…”

mentioning

confidence: 99%

Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

Phelps

Lee

Jose

et al. 2013

122

DNA "breathing" is a thermally driven process in which basepaired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-μs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.smFRET | single-molecule linear dichroism | thermal fluctuations | T4 primosome helicase | DNA helicase D NA "breathing" is defined as the transient opening, due to thermal fluctuations, of nucleic acid base pairs at experimental temperatures below the melting temperature of dsDNA duplex. Such fluctuations are thought to be important to the function of replication, transcription, recombination, and repair systems, which depend on the ability of the relevant protein complexes to gain access to ssDNA templates that are located in the dsDNA "interior" (1-3). Furthermore, the ability of the genome to spontaneously expose partial template sequences is a central feature of protein-DNA binding models in which open conformations serve as substrates that can be "trapped" by a functional protein complex. McConnell and von Hippel (4) suggested that breathing of duplex DNA might be structurally decomposed into a set of distinct breathing elements, each involving characteristic fluctuations of the sugar-phosphate backbone and the nucleobases. For example, a possible motion might involve compression along the double-helix axis (perhaps coupled with twisting or bending), leading to the disruption of WatsonCrick hydrogen bonds without...

“…Although MyoV is among the most extensively studied motor proteins, improvements in experimental resolution continue to provide new and surprising insights into the details of its dynamics. A beautiful recent example is the high-speed atomic force microscopy (AFM) of Kodera et al (20), which was used to visualize not only the expected hand-over-hand stepping but additional, less wellunderstood processes like "foot stomping" (21,22), where one head detaches and rebinds to the same site. Thus, a comprehensive picture of MyoV motility needs to account for all the kinetic pathways, including back-stepping and foot stomping, how they vary under load, and their relationship to the structural and chemical parameters of the motor.…”

mentioning

confidence: 99%

“…A beautiful recent example is the high-speed atomic force microscopy (AFM) of Kodera et al (20), which was used to visualize not only the expected hand-over-hand stepping but additional, less wellunderstood processes like "foot stomping" (21, 22), where one head detaches and rebinds to the same site. Thus, a comprehensive picture of MyoV motility needs to account for all the kinetic pathways, including back-stepping and foot stomping, how they vary under load, and their relationship to the structural and chemical parameters of the motor.To address these issues, we introduce a minimal model of MyoV dynamics, focusing on the stochastic fluctuations of the motor head during the diffusive search of the detached head for a binding site, whose importance has been illuminated by various experiments (22)(23)(24)(25). The large persistence length l p of the lever arms (26-28) allows us to propose a coarse-grained polymer model for the reaction-diffusion problem, which, in turn, yields approximate analytical expressions for all the physical observables, including binding times, run length, velocity, and stall force.…”

mentioning

confidence: 99%

“…To address these issues, we introduce a minimal model of MyoV dynamics, focusing on the stochastic fluctuations of the motor head during the diffusive search of the detached head for a binding site, whose importance has been illuminated by various experiments (22)(23)(24)(25). The large persistence length l p of the lever arms (26)(27)(28) allows us to propose a coarse-grained polymer model for the reaction-diffusion problem, which, in turn, yields approximate analytical expressions for all the physical observables, including binding times, run length, velocity, and stall force.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Design principles governing the motility of myosin V

Hinczewski

Tehver

Thirumalai

2013

The molecular motor myosin V (MyoV) exhibits a wide repertoire of pathways during the stepping process, which is intimately connected to its biological function. The best understood of these is the hand-over-hand stepping by a swinging lever arm movement toward the plus end of actin filaments. Single-molecule experiments have also shown that the motor "foot stomps," with one hand detaching and rebinding to the same site, and back-steps under sufficient load. The complete taxonomy of MyoV's load-dependent stepping pathways, and the extent to which these are constrained by motor structure and mechanochemistry, are not understood. Using a polymer model, we develop an analytical theory to describe the minimal physical properties that govern motor dynamics. We solve the first-passage problem of the head reaching the target-binding site, investigating the competing effects of backward load, strain in the leading head biasing the diffusion in the direction of the target, and the possibility of preferential binding to the forward site due to the recovery stroke. The theory reproduces a variety of experimental data, including the power stroke and slow diffusive search regimes in the mean trajectory of the detached head, and the force dependence of the forward-to-backward step ratio, run length, and velocity. We derive a stall force formula, determined by lever arm compliance and chemical cycle rates. By exploring the MyoV design space, we predict that it is a robust motor whose dynamical behavior is not compromised by reasonable perturbations to the reaction cycle and changes in the architecture of the lever arm.functional robustness | polymer physics | architectural basis | stall force expression M yosin V (MyoV), a cytoskeletal motor protein belonging to the myosin superfamily (1), converts energy from ATP hydrolysis into the transport of intracellular cargo, such as mRNA and organelles along actin filaments (2). In its dimeric form, the motor has two actin-binding, ATPase heads connected to α-helical lever arm domains stiffened by attached calmodulins or essential light chains (Fig. 1). The nucleotide-driven mechanochemical cycle of the heads produces two changes in the lever arm orientation: a power stroke, where an actin-bound head swings the lever arm forward toward the plus (barbed) end of the filament, and a recovery stroke, which returns the arm to its original configuration when the head is detached from actin (3). The motor translates these changes into processive plus end-directed movement (4-6). By alternating head detachment, MyoV walks hand-over-hand (7,8), taking one step of ≈36 nm for each ATP consumed (9). At small loads, the motor can complete ≈20 − 60 forward steps before dissociating from actin (6, 10, 11). Such a high unidirectional processivity requires coordination in the detachment of the two heads, a "gating" mechanism, which is believed to arise from the strain within the molecule when both heads are bound to actin (12-15). Sufficiently large opposing loads can counteract the plus end-directed ...