Different degrees of lever arm rotation control myosin step size

Myosins are actin-based motors that are generally believed to move by amplifying small structural changes in the core motor domain via a lever arm rotation of the light chain binding domain. However, the lack of a quantitative agreement between observed step sizes and the length of the proposed lever arms from different myosins challenges this view. We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins. Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain. The extrapolated pivoting point resided in the same region of the rat Myo1d head domain as in conventional myosins. Therefore, rat Myo1d achieves its larger working stroke by a large calculated approximately 90 degrees rotation of the light chain binding domain. These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.     

Flexibility of the drinking mechanism in adult chickens (Gallus gallus) (Aves)

Summary Drinking was studied in adult chickens by cinematography and radiography. Three subsequent behavioral phases occur. (1) Water is transported from the water box into the oropharynx when the beak is immersed. Then delicately tuned cyclic motion patterns of beaks, tongue, and larynx transport water by capillarity, squeezing, and suction. (2) During the elevation of the head the tongue is elevated and the larynx is depressed to keep the water in the pharynx against the gravitational and centrifugal forces that result from the upward swing of the head. (3) During the tip up phase gravity transports the water to the esophagus, while the adhering water is pushed and squeezed caudad by tongue and larynx movements. Flexibility in the adult drinking mechanism was analyzed by comparing the drinking of normal and beak-trimmed chickens under normal drinking conditions, as well as while drinking small drops. Three modes of behavioral flexibility were discussed: conservative, regressive, and progressive flexibility. Most behavioral elements of the modal action pattern in drinking are so flexible that a chicken can reorganize the movement patterns of jaws, tongue, larynx, and head to adapt the mechanism to external (drop drinking) or internal (beak-trimming) changes. However, in drop drinking, the normal chicken relies upon a regressive takeover by an ontogenetically earlier developed pattern in the craniocervical motion system. Presence of the observed progressive flexibility in lingual and cervical motion patterns is shown to be a precondition for the avian drinking mechanism to keep up with dominant evolutionary changes in feeding mechanisms.     

Myosin motor domain lever arm rotation is coupled to ATP hydrolysis

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.     

Conformational selection during weak binding at the actin and myosin interface

The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF(4), ADP-BeF(x), and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.     

The position of the rotation center of the glenohumeral joint

To validate the assumption that the center of rotation in the glenohumeral (GH) joint can be described based on the geometry of the joint, two methods for calculation of the GH rotation center were compared. These are a kinematic estimation based on the calculation of instantaneous helical axes, and a geometric estimation based on a spherical fit through the surface of the glenoid. Four fresh cadaver arms were fixed at the scapula and fitted with electromagnetic sensors. Each arm was moved in different directions while at the same time the orientation of the humerus was recorded. Subsequently, each specimen was dissected and its glenoid and humeral head surfaces were digitized. Results indicate no differences between the methods. It is concluded that the method to estimate the GH center of rotation as the center of a sphere through the glenoid surface, with the radius of the humeral head, appears to be valid.     

Cooperativity of myosin molecules through strain-dependent chemistry

There is mounting evidence that the myosin head domain contains a lever arm which amplifies small structural changes that occur at the nucleotide-binding site. The mechanical work associated with movement of the lever affects the rates at which the products of ATP hydrolysis are released. During muscle contraction, this strain-dependent chemistry leads to cooperativity of the myosin molecules within a thick filament. Two aspects of cooperative action are discussed, in the context of a simple stochastic model. (i) A modest motion of the lever arm on ADP release can serve to regulate the fraction of myosin bound to the thin filament, in order to recruit more heads at higher loads. (ii) If the lever swings through a large angle when phosphate is released, the chemical cycles of the myosin molecules can be synchronized at high loads. This leads to stepwise sliding of the filaments and suggests that the isometric condition is not a steady state.     

Exploration of the conformational space of myosin recovery stroke via molecular dynamics

Muscle contractions are driven by cyclic conformational changes of myosin, whose molecular mechanisms of operation are being elucidated by recent advances in crystallographic studies and single molecule experiments. To complement such structural studies and consider the energetics of the conformational changes of myosin head, umbrella sampling molecular dynamics (MD) simulations were performed with the all-atom model of the scallop myosin sub-fragment 1 (S1) with a bound ATP in solution in explicit water using the crystallographic near-rigor and transition state conformations as two references. The constraints on RMSD reaction coordinates used for the umbrella sampling were found to steer the conformational changes efficiently, and relatively close correlations have been observed between the set of characteristic structural changes including the lever arm rotation and the closing of the nucleotide binding pocket. The lever arm angle and key residue interaction distances in the nucleotide binding pocket and the relay helix show gradual changes along the recovery stroke reaction coordinate, consistent with previous crystallographic and computational minimum energy studies. Thermal fluctuations, however, appear to make the switch-2 coordination of ATP more flexible than suggested by crystal structures. The local solvation environment of the fluorescence probe, Trp 507 (scallop numbering), also appears highly mobile in the presence of thermal fluctuations.     

Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence

Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the cross-bridge attached to actin. To elucidate cross-bridge structure during force development, two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from two extrinsic probes. The probes were located at two interfaces containing the catalytic, converter, and lever arm domains of S1. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Best agreement between the computed and observed signals gave structures showing that actin binding to S1 causes movement of the lever arm. A three-state model of S1 conformation during contraction consists of three actin-bound cross-bridge states observed from muscle fibers in isometric contraction, in the presence of MgADP, and in rigor. Structures best representing these states show that most of the lever arm rotation occurs between isometric contraction and the MgADP states, i.e., during phosphate release. Smaller but significant lever arm rotation occurs with ADP dissociation. Structural changes within the S1 interfaces studied are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4834-4843].     

Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin

The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the ~270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by ~20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.     

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

Changes in axial stiffness of the trunk as a function of walking speed

Research suggests that abnormal coordination patterns between the thorax and pelvis in the transverse plane observed in patients with Parkinson's disease and the elderly might be due to alteration in axial trunk stiffness. The purpose of this study was to develop a tool to estimate axial trunk stiffness during walking and to investigate its functional role. Fourteen healthy young subjects participated in this study. They were instructed to walk on the treadmill and kinematic data was collected by 3D motion analysis system. Axial trunk stiffness was estimated from the angular displacement between trunk segments and the amount of torque around vertical axis of rotation. The torque due to arm swing cancelled out the torque due to the axial trunk stiffness during walking and the thoracic rotation was of low amplitude independent of changes in walking speeds within the range used in this study (0.85-1.52 m/s). Estimated axial trunk stiffness increased with increasing walking speed. Functionally, the suppression of axial rotation of thorax may have a positive influence on head stability as well as allowing recoil between trunk segments. Furthermore, the increased stiffness at increased walking speed would facilitate the higher frequency rotation of the trunk in the transverse plane required at the higher walking speeds.     

Cervical spine motion in the sagittal plane: Kinematic and geometric parameters

The kinematic function of the cervical spine has been examined previously by means of cineradiography or a sequence of lateral X-rays, usually of a flexion-extension range of motion. Interpretation of these studies, however, presents difficulties. One of the major problems is how to extract information from the X-ray images which is not only explicit, quantitative and accurate, but which is also diagnostically useful. Another problem is that as one increases the number of steps of motion between full flexion and full extension to obtain a more detailed examination, one also increases the amount of radiation exposure and the bulk of the data.

Reported here is a technique which uses one lateral-view X-ray for each of five neck positions: full flexion, full extension, and three intermediate positions. From each set of X-rays, various parameters are derived to describe two types of data: kinematic (angles and centers of rotation), and geometric (pattern of curvature). This technique has been shown capable of identifying functional abnormalities in patients with neck pain who have no structural abnormalities detectable by X-ray. Further experience will be useful in better defining various types of functional abnormalities.     

Cervical helical axis characteristics and its center of rotation during active head and upper arm movements-comparisons of whiplash-associated disorders, non-specific neck pain and asymptomatic individuals

The helical axis model can be used to describe translation and rotation of spine segments. The aim of this study was to investigate the cervical helical axis and its center of rotation during fast head movements (side rotation and flexion/extension) and ball catching in patients with non-specific neck pain or pain due to whiplash injury as compared with matched controls. The aim was also to investigate correlations with neck pain intensity. A finite helical axis model with a time-varying window was used. The intersection point of the axis during different movement conditions was calculated. A repeated-measures ANOVA model was used to investigate the cervical helical axis and its rotation center for consecutive levels of 15 degrees during head movement. Irregularities in axis movement were derived using a zero-crossing approach. In addition, head, arm and upper body range of motion and velocity were observed. A general increase of axis irregularity that correlated to pain intensity was observed in the whiplash group. The rotation center was superiorly displaced in the non-specific neck pain group during side rotation, with the same tendency for the whiplash group. During ball catching, an anterior displacement (and a tendency to an inferior displacement) of the center of rotation and slower and more restricted upper body movements implied a changed movement strategy in neck pain patients, possibly as an attempt to stabilize the cervical spine during head movement.     

CaATP as a substrate to investigate the myosin lever arm hypothesis of force generation

In an effort to test the lever arm model of force generation, the effects of replacing magnesium with calcium as the ATP-chelated divalent cation were determined for several myosin and actomyosin reactions. The isometric force produced by glycerinated muscle fibers when CaATP is the substrate is 20% of the value obtained with MgATP. For myosin subfragment 1 (S1), the degree of lever arm rotation, determined using transient electric birefringence to measure rates of rotational Brownian motion in solution, is not significantly changed when calcium replaces magnesium in an S1-ADP-vanadate complex. Actin activates S1 CaATPase activity, although less than it does MgATPase activity. The increase in actin affinity when S1. CaADP. P(i) is converted to S1. CaADP is somewhat greater than it is for the magnesium case. The ionic strength dependence of actin binding indicates that the change in apparent electrostatic charge at the acto-S1 interface for the S1. CaADP. P(i) to S1. CaADP step is similar to the change when magnesium is bound. In general, CaATP is an inferior substrate compared to MgATP, but all the data are consistent with force production by a lever arm mechanism for both substrates. Possible reasons for the reduced magnitude of force when CaATP is the substrate are discussed.     

Elastic lever-arm model for myosin V

We present a mechanochemical model for myosin V, a two-headed processive motor protein. We derive the properties of a dimer from those of an individual head, which we model both with a four-state cycle (detached; attached with ADP.Pi; attached with ADP; and attached without nucleotide) and alternatively with a five-state cycle (where the powerstroke is not tightly coupled to the phosphate release). In each state the lever arm leaves the head at a different, but fixed, angle. The lever arm itself is described as an elastic rod. The chemical cycles of both heads are coordinated exclusively by the mechanical connection between the two lever arms. The model explains head coordination by showing that the lead head only binds to actin after the powerstroke in the trail head and that it only undergoes its powerstroke after the trail head unbinds from actin. Both models (four- and five-state) reproduce the observed hand-over-hand motion and fit the measured force-velocity relations. The main difference between the two models concerns the load dependence of the run length, which is much weaker in the five-state model. We show how systematic processivity measurement under varying conditions could be used to distinguish between both models and to determine the kinetic parameters.     

Static and dynamic assessment of the Biodex dynamometer

The validity and accuracy of the Biodex dynamometer was investigated under static and dynamic conditions. Static torque and angular position output correlated well with externally derived data (r = 0.998 and r greater than 0.999, respectively). Three subjects performed maximal voluntary knee extensions and flexions at angular velocities from 60 to 450 degrees.s-1. Using linear accelerometry, high speed filming and Biodex software, data were collected for lever arm angular velocity and linear accelerations, and subject generated torque. Analysis of synchronized angular position and velocity changes revealed the dynamometer controlled angular velocity of the lever arm to within 3.5% of the preset value. Small transient velocity overshoots were apparent on reaching the set velocity. High frequency torque artefacts were observed at all test velocities, but most noticeably at the faster speeds, and were associated with lever arm accelerations accompanying directional changes, application of resistive torques by the dynamometer, and limb instability. Isokinematic torques collected from ten subjects (240, 300 and 400 degrees.s-1) identified possible errors associated with reporting knee extension torques at 30 degrees of flexion. As a result of tissue and padding compliance, leg extension angular velocity exceeded lever arm angular velocity over most of the range of motion, while during flexion this compliance meant that knee and lever arm angles were not always identical, particularly at the start of motion. Nevertheless, the Biodex dynamometer was found to be both a valid and an accurate research tool; however, caution must be exercised when interpreting and ascribing torques and angular velocities to the limb producing motion.     

Scapular kinematics during unloaded and maximal loaded isokinetic concentric and eccentric shoulder flexion and extension movements

Characterization of scapular kinematics under demanding load conditions might aid to distinguish between physiological and clinically relevant alterations. Previous investigations focused only on submaximal external load situations. How scapular movement changes with maximal load remains unclear. Therefore, the present study aimed to evaluate 3D scapular kinematics during unloaded and maximal loaded shoulder flexion and extension. Twelve asymptomatic individuals performed shoulder flexion and extension movements under unloaded and maximal concentric and eccentric loaded isokinetic conditions. 3D scapular kinematics assessed with a motion capture system was analyzed for 20° intervals of humeral positions from 20° to 120° flexion. Repeated measures ANOVAs were used to evaluate kinematic differences between load conditions for scapular position angles, scapulohumeral rhythm and scapular motion extent. Increased scapular upward rotation was seen during shoulder flexion and extension as well as decreased posterior tilt and external rotation during eccentric and concentric arm descents of maximal loaded compared to unloaded conditions. Load effects were further seen for the scapulohumeral rhythm with greater scapular involvement at lower humeral positions and increased scapular motion extent under maximal loaded shoulder movements. With maximal load applied to the arm physiological scapular movement pattern are induced that may imply both impingement sparing and causing mechanisms.     

Myosin II isoforms in smooth muscle: heterogeneity and function

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC(20) has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ.     

Can the effect of soft tissue artifact be eliminated in upper-arm internal-external rotation?

The purpose of this study was to quantify the effect of soft tissue artifact during three-dimensional motion capture and assess the effectiveness of an optimization method to reduce this effect. Four subjects were captured performing upper-arm internal-external rotation with retro-reflective marker sets attached to their upper extremities. A mechanical arm, with the same marker set attached, replicated the tasks human subjects performed. Artificial sinusoidal noise was then added to the recorded mechanical arm data to simulate soft tissue artifact. All data were processed by an optimization model. The result from both human and mechanical arm kinematic data demonstrates that soft tissue artifact can be reduced by an optimization model, although this error cannot be successfully eliminated. The soft tissue artifact from human subjects and the simulated soft tissue artifact from artificial sinusoidal noise were demonstrated to be considerably different. It was therefore concluded that the kinematic noise caused by skin movement artifact during upper-arm internal-external rotation does not follow a sinusoidal pattern and cannot be effectively eliminated by an optimization model.