Single molecule mechanics resolves the earliest events in force generation by cardiac myosin

Michael S. Woody, Donald A. Winkelmann, Marco Capitanio, E. Michael Ostap, Yale E. Goldman

Preprint posted on 27 June 2019

Article now published in eLife at

Caught in the act: measuring cardiac myosin's catalytic cycle at high spatial and temporal resolution

Selected by Alyson Smith

Categories: biochemistry, biophysics

Why I think this study is interesting

Myosin uses a cycle of ATP hydrolysis coupled to conformational changes to pull on actin filaments and sustain eukaryotic life. This study uses ultra-fast force clamping, a sensitive biophysical assay, to capture the fastest events in myosin’s ATPase cycle, which have remained opaque to previous assays. The study’s results provide insight into the inner workings of the myosin motor and can pinpoint problems underlying human diseases caused by myosin mutations, such as cardiomyopathy, muscular dystrophy, or cancer.


Most of the dozens of myosin isoforms—including skeletal, cardiac, smooth muscle, and nonmuscle—follow the force-generating cycle diagrammed below (1). First (steps A and B), myosin motor domain heads hydrolyze ATP to ADP and inorganic phosphate (Pi). At this stage, myosin heads bind weakly to actin filaments. Next (step C), myosin releases phosphate. At around the same time, myosin’s lever arm swings to produce the force-generating power stroke (termed working stroke in this preprint). Finally (step D), myosin heads exchange ADP for ATP and release actin to restart the cycle.

The myosin ATPase cycle. From Kaplinsky and Mallarkey 2018.


Following actin binding by myosin motor domains (step B), conformational changes in myosin and actin, the swinging of the lever arm, and phosphate release occur within milliseconds, making each event difficult to resolve with conventional biophysical methods. This study zooms in on these events to determine which occurs first for human beta-cardiac myosin: the power stroke or phosphate release.

To achieve high temporal resolution, the authors used an ultra-fast force clamp with a three-bead optical trap (2). Two laser-controlled beads moved an actin filament across a coverslip holding myosin molecules attached to beads. When the actin encountered a myosin head, force feedback loops applied to the beads detected sub-nanometer displacements of the actin at intervals of less than 100 microseconds.

The three-bead optical trap used in ultra-fast force clamp experiments. From Finer JT, Simmons, RM, and Spudich JA, 1994.


The high resolution of ultra-fast force clamping produced traces of myosin-actin interactions filled with fluctuations due to Brownian motion. This variability precluded applying computational techniques (step finding algorithms, hidden Markov chains, or Bayesian non-parametric analysis) to individual traces. The authors instead used ensemble averages of many traces, aligned at the beginning of the myosin-actin interactions, to detect the power stroke and other events in their experiments.

Key findings

Many myosin-actin interactions are short-lived

Many myosin-actin interactions ended before myosin completed its ATPase cycle. The frequency of these short-lived interactions increased with applied load; up to 50% lasted less than 10 milliseconds at the highest load (4.5 picoNewtons). Despite their transience, short-lived interactions stopped the laser-powered motion of the actin and held it for hundreds of microseconds. The direction of the applied load affected the relationship between force and interaction duration. This polarity indicates that the short-lived state represents a weak but stereospecific interaction between actin and myosin that either ends quickly or proceeds to a strong-binding state and force generation.

The power stroke occurs before phosphate release

The ultra-fast force clamp detected myosin’s power stroke as a small displacement that occurred within 5 milliseconds of actin binding. The stroke happened faster than both myosin-actin detachment and phosphate release, making it unlikely that phosphate release occurs before the power stroke. Ten millimolar free phosphate did not change the stroke rate, further supporting the author’s model that the power stroke occurs before phosphate release.

The power stroke and phosphate release are reversible

After the power stroke reached its peak actin displacement, the authors saw small displacements in the opposite direction. Adding free phosphate to the assay amplified this reversal and decreased the final displacement. This displacement reversal indicates that free phosphate can re-bind after release, slowing myosin’s ATPase cycle. The fact that free phosphate affects post-stroke displacement reversal but not the power stroke itself further supports the author’s hypothesized order of events: power stroke first and phosphate release second.

Figure 6 from this preprint. Proposed model with short-lived, weakly-bound attachments directly preceding the working stroke, which is followed by phosphate release and potential phosphate rebinding.


Future directions

According to the author’s data and simulations, myosin remains bound to actin in a pre-stroke state for ~300 microseconds before a stroke reversal but ~1 millisecond following a reversal. This increased state duration suggests that myosin does not return to its original conformation following phosphate re-binding and stroke reversal. Future simulations and biophysical experiments could test this hypothesis.

The results of this study agree with previous work on skeletal and cardiac myosin, non-muscle myosin V, and ultra-fast force clamp experiments using fast-skeletal muscle myosin (see references in preprint). The dozens of eukaryotic myosin isoforms differ in sequence, structure, kinetics, and biomechanics (3). Further research using ultra-fast force clamps or other sensitive biophysical techniques may reveal different mechanisms for myosin force generation.

How do mutations in the myosin motor domain affect the power stroke and phosphate release? Disease-causing mutations can occur in the actin-myosin interface, in the ATP/ADP binding site of myosin, or in myosin domains required to support conformational changes. Ultra-fast force clamp assays could determine how these mutations affect the power stroke and phosphate release and whether potential therapeutics lessen their effects.


1. Kaplinsky E and Mallarkey G. Cardiac myosin activators for heart failure therapy: focus on omecamtiv mecarbil. Drugs Context 7:212518 (2018).

2. Finer JT, Simmons, RM, and Spudich JA. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119 (1994).

3. Sweeney HL and Houdusse A. Structural and functional insights into the Myosin motor mechanism. Annu Rev Biophys 39:539-557 (2010).

Tags: enzyme kinetics, molecular motor, myosin, optical trapping, power stroke

Posted on: 21 August 2019


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