Direct Observation of the Myosin Va Recovery Stroke That Contributes to Unidirectional Stepping along Actin

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed “strokes”; the “power stroke” is the force-generating swinging of the myosin light chain–binding “neck” domain relative to the motor domain “head” while bound to actin; the “recovery stroke” is the necessary initial motion that primes, or “cocks,” myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a “hand over hand” mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥5 kBT of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va

Direct Observation of Strand Passage by DNA- Topoisomerase and Its Limited Processivity

Abstract Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIa, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.5160.33 mm (1166 turns) of a braid was unlinked in a burst of reactions taking 864 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ,100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ,10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ,10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments

Distribution of unbraiding burst sizes.

<p>(<b>A</b>) The expected length of unbraiding. If a topo IIα molecule binds at <i>i</i>-th turn (circle) from an end of a braid of <i>n</i> turns, and if the topo IIα stays and remains active for a sufficient period, then the braid length will become <i>n</i> – 2<i>i</i>, or the unbraiding length will be 2<i>i</i>, because the two DNA segments forming the braid can freely slide against each other to keep the braid center at the same position. For random binding, <i>i</i> is anywhere between 0 and <i>n</i>/2, and thus the unbraiding length 2<i>i</i> will distribute equally between 0 and <i>n</i>, averaging <i>n</i>/2. Thermal motion of DNA will increase the unbraiding length by several turns (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034920#pone.0034920.s006" target="_blank">Text S1</a>). (<b>B</b>) Observed distribution. The unbraiding lengths in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034920#pone-0034920-g002" target="_blank">Figure 2A</a> are normalized by the length before unbraiding (corresponding to <i>n</i> in <b>A</b>). Data with an initial length greater than 1 µm have been selected and analyzed. The leftmost bars with stripes represent cases where unbraiding was undetected in the length assay; the normalized unbraiding lengths for these data should be less than 0.25 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034920#pone.0034920.s004" target="_blank">Figure S4</a>).</p

Unbraiding burst sizes.

<p>(<b>A</b>) Burst sizes estimated from the braid length in fluorescence images. (<b>B</b>) Burst sizes in terms of braid turns; the braid length was converted to braid turns using the calibration equation described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034920#pone.0034920.s003" target="_blank">Figure S3</a>. For both panels, dots show individual observations, plotted in two or three lines for clarity. Large circles show averages for the first (blue or purple), second (green), and third (cyan) bursts, counterclockwise (CCW) and clockwise (CW) braids not distinguished. The averages are for the data in which unbraiding was confirmed as a change in braid length (dots in the central white zone). Dots in the top shaded zone indicate cases where a braid completely disappeared by the time the tension was set up (time 0). The shaded zone below vertical zero shows cases where a length change was undetected but mechanical unwinding at the end (300 s) revealed remaining braid turns of less than 30. The bottom shaded zone is for no reaction cases where the braid number remained 30 until 300 s as confirmed by mechanical unwinding. Insets at top right show histograms of unbraided length/turns at 3.7 pM topo IIα; <i>ud</i>, undetected; <i>nr</i>, no reaction.</p

Experimental design.

<p>(<b>A</b>) Schematic diagrams showing the experimental procedure where the floating ends of the two DNAs were manipulated with optical tweezers while their root positions were controlled by stage movement. Numbers correspond to those in <b>B</b>–<b>D</b>. (<b>B</b>) Snapshots of bright-field (1–3; sequential frames) and fluorescence images (4–12; averaged over 30 frames = 1 s). Arrows in 1–3 show the direction of stage movement. Rectangles in 9 show the regions where the DNA images were fitted with a line to estimate the braid length. The scale bar in 12 shows 5 µm (57.5 pixels). (<b>C, D</b>) Time courses of the braid length (<b>C</b>) and the DNA tension sensed by the lower-left bead in <b>B</b> (<b>D</b>). Time 0 is the end of stage movement. After the processive unbraiding at ∼60 s, we slightly increased the tension at ∼70 s. Gray dots in <b>C</b> were calculated on images averaged over 30 frames, and further averaging over 120 frames (4 s) shown in blue. Red broken lines show the way the burst time was estimated. Green horizontal bars in <b>C</b> indicate portions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034920#pone.0034920.s005" target="_blank">Viedo S1</a>. The total tension in <b>D</b> represents (<i>T</i>_x²+<i>T</i>_y²)^1/2, and thus noise, converted to positive values, dominates over the actual tension when the latter is below the noise level. The tension is essentially zero at stages 4 and 12.</p

Mechanically driven ATP synthesis by F-1-ATPase

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction(1-3). The F-1 portion of ATP synthase, also known as F-1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates(4) against the surrounding alpha(3)beta(3) subunits(5), hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F-o portion of ATP synthase, leads to ATP synthesis in biological systems(1-3,6,7). Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F-1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force ( torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium