Kinesin hydrolyses one ATP per 8-nm step. Mark J. Schnitzer *† & Steven M. Block†‡ Departments of * Physics and † Molecular Biology, and ‡ Princeton Materials Institute, Princeton University, Princeton, New Jersey 08544, USA Nature 24 Juli 1997, vol. 388, pp. 386-390. Basics. Kinesin :
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Mark J. Schnitzer*† & Steven M. Block†‡
Departments of * Physics and † Molecular Biology, and ‡ Princeton Materials Institute, Princeton University, Princeton, New Jersey 08544, USA
Nature 24 Juli 1997, vol. 388, pp. 386-390
Two-headed ATP driven motor protein that moves along the microtubules in discrete steps of 8 nm.
How many molecules of ATP are consumed per step?
Using the processivity of kinesin, statistical analysis of intervals between steps at limiting ATP and studies of fluctuations in motor speed as a function of [ATP]
Silica beads (0.5 mm) with nonspecifically bound kinesin captured in an optical trap and deposited onto immobilized microtubules bound to the coverglass. Subsequent movements recorded with optical-trapping interferometry.
Low concentrations of kinesin protein ensures maximum one kinesin per bead.
Applied force: 7 fN nm-1 => mean force < 0.9 pN (< 15 % stall)
yielded kcat = 680 ± 31 nms-1and Km = 62 ± 5 mM.
Poisson statistics f = 1 - exp(-lC) confirms that single molecules suffice to move beads. (c2 = 0.7)
Advance of kinesin molecules in clear increments of 8 nm (minority of other step sizes cannot be excluded).
At limiting [ATP] kcat/Km = 11 ± 1 nms-1 mM-1implying stepping rate of 1.4 ± 0.1 mM-1 s-1
Exponential distribution => solitary, rate-limiting biochemical reaction (ATP binding) i.e. kinesin requires only one ATP per step.
If (n) ATP molecules were needed the distribution would be a convolution of n exponentials.
Data well fit by single exponential (reduced c2 = 0.6) with a rate of 1.1 ± 0.1 mM-1 s-1
(Two exponentials gave c2 = 1.4 with a rate of 0.8 ± 0.06 mM-1 s-1)
For single processive motors, fluctuations about the average speed reflect underlying enzyme stochasticity.
Randomness parameter, r, is a dimensionless measure of the temporal irregularity between steps. For motors that step a distance, d, and whose positions are functions of time, x(t), it’s:
Since both numerator and denominator increase linearly in time, their ratio approaches a constant. The reciprocal of this constant, r-1, supplies a continuous measure of the number of rate-limiting transitions per step.
Robust to sources of thermal and instrumental noise and without need to identify individual stepwise transitions.
Test of determinability of r performed with both simulated and real data.
Simulated: Stochastic staircase records and gaussian white noise.
Real: 2 mM AMP-PNP (non-hydrolysable ATP analogue) and movement of stage.
Both tests positive i.e. stepping statistics could be distinguished.
Saturating ATP value implies minimum two rate-limiting transitions per step. Biochemical pathways also predict r ≈ ½ with assumption of one hydrolysis per step.
For limiting [ATP], r rises through 1, reflecting a single rate-limiting transition once per advance (ATP binding).
Why is r > 1 at limiting [ATP]?
Not heterogeneity in ATP binding rate, bead size or stiffness of bead-kinesin linkage, futile hydrolysis, sticking or transient inactive states.
Maybe backwards movement (7 %) and/or double step (16 %).
Kinesin hydrolyses only one ATP per 8 nm step. Models consistent with this result is:
Another future challenge lies in understanding the molecular basis of kinesin movement, since the motor domain of kinesin is quite small (4.5 x 4.5 x 7.0 nm) compared to the 8 nm step.