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1.
Figure 5

Figure 5. From: Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers.

Eg5-513-His velocity, v, versus ADP concentration (0–2 mM; N = 10–207) measured at three different ATP concentrations at F = −3.0 ± 0.1 pN.

Megan T. Valentine, et al. Biophys J. 2009 September 16;97(6):1671-1677.
2.
Figure 4

Figure 4. From: Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers.

Eg5-513-His velocity, v, versus ATP concentration with (open circles; N = 20–209) and without 10 mM Pi (solid circles; N = 7–200) for F = −3.0 ± 0.1 pN. Solid and dashed lines are fits to a competitive inhibition model, with constant KI = 6 ± 2 mM. (Inset) Velocity and randomness for 2000 μM (circles; N = 60–212), 500 μM (squares; N = 44–115), and 100 μM (triangles; N = 91–150) versus phosphate concentration.

Megan T. Valentine, et al. Biophys J. 2009 September 16;97(6):1671-1677.
3.
Figure 3

Figure 3. From: Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers.

Eg5 processivity as a function of ADP and Pi concentrations. (A) Run length L (lower) and the dwell time ratio for the last and preceding steps (upper) does not show a clear trend with Pi concentration for [ATP] = 100 μM and [ATP] = 2000 μM (N = 96–212). (B) Run length L (lower) and the ratio of the average dwell time for the last and preceding steps (upper) both decrease with ADP concentration (N = 10–207). The processivity is also ATP-dependent: L decreased by ∼50% when [ADP] = [ATP].

Megan T. Valentine, et al. Biophys J. 2009 September 16;97(6):1671-1677.
4.
Figure 2

Figure 2. From: Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers.

Eg5 processivity as a function of ATP concentration and force. (A) Run length, L (lower), as a function of [ATP] for F = 0 pN (circles; N = 31–71 runs at each condition,), F = −3.0 ± 0.1 pN (triangles; N = 20–207), and F = −3.8 ± 0.1 pN (squares; N = 44–110). Run length was nearly independent of [ATP] for all forces. The ratio of the average dwell time for the last and preceding steps (upper) remains near unity over three orders of magnitude in ATP concentration (F = −3.8 pN). (B) Run length L (lower) as a function of force for [ATP] = 2000 μM (circles; N = 11–277) and [ATP] = 16 μM (squares; N = 72–97). L decreases weakly with the magnitude of applied force, for both assisting and hindering loads. The ratio of the average dwell time for the last and preceding steps (upper) remains near unity for |F| < ∼4 pN, but decreases for forces beyond this value.

Megan T. Valentine, et al. Biophys J. 2009 September 16;97(6):1671-1677.
5.
Figure 1

Figure 1. From: Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers.

A representative data record showing the processive stepping of a single Eg5-513-His dimer versus time. The median-filtered bead position (25-point window, dark trace) is superimposed on the unfiltered position (light gray trace), and displays clear stepwise transitions (vertical lines) and the final dissociation event. Horizontal gridlines are spaced at 8 nm to indicate the approximate molecular step size. The dwell times for steps (except the last) in all records, τi, were recorded and averaged as 〈τ〉; the dwell times for the final steps were separately recorded and averaged as 〈τlast〉. (Inset) Cartoon representation of the experimental geometry (not to scale). An Eg5-513-His homodimer is attached to a bead held in an optical trap as it walks along the MT under force-clamped conditions.

Megan T. Valentine, et al. Biophys J. 2009 September 16;97(6):1671-1677.
6.
Figure 6

Figure 6. From: Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers.

A five-state kinetic scheme (S0–S5) for Eg5 stepping during a processive run, in which Eg5-microtubule dissociation occurs from a one-head-bound state before ATP binding (S3). The model is consistent with single-molecule and solution biochemical data for human Eg5-513 dimers (6). State S0 occurs immediately after the rapid translocation step, the starting point for the cycle, which is easily identified in single-molecule records. The trailing head binds ATP and the advancing head binds ADP, until it forms a tight connection to the forward binding site on the MT. In states S0 and S1, there is a rapid equilibrium at the leading head between the ADP-bound and no-nucleotide states, indicated by the blue-and-white coloring; arrows indicate the resultant transition between weak and strong MT binding. The neck linker of the trailing head is docked (heavy black line). Hydrolysis by the trailing head in S1 is followed by complete ADP release from the leading head in S2. Then, phosphate is released from the trailing head to form the ATP waiting state (S3), in which one head is bound to the MT while its partner is free. Either the premature binding of ADP to the MT-bound head or the application of large hindering or assisting loads can promote MT dissociation from this state. In this model, dissociation competes with ATP binding to the MT-bound head (S4). Successful ATP binding leads to a subsequent forward translocation, restarting the mechanochemical cycle. The new starting state (S5) is biochemically and structurally identical to the original (S0), but the centroid of the motor has now advanced by ∼8 nm along the MT lattice.

Megan T. Valentine, et al. Biophys J. 2009 September 16;97(6):1671-1677.

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