Kinesin Takes One 8-nm Step for Each ATP That It Hydrolyzes

Abstract

Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1.08 ؎ 0.09 steps for each ATP hydrolyzed at 1 mM ATP. Therefore, under low loads in which the drag force < < 1 pN, coupling between the chemical and mechanical cycles of kinesin is tight, consistent with conventional power stroke models. Our results rule out models that require two or more ATPs/step, such as some thermal ratchet models, or that propose multiple steps powered by single ATPs.Conventional kinesin is a protein machine that steps along the surface of a microtubule as it carries a membrane-bound organelle toward the periphery of a cell (1-3). The size of the steps is ϳ8 nm (4,5). This is the distance between consecutive binding sites along the microtubule protofilament (6, 7), and a single kinesin molecule can take hundreds of steps without detaching (8, 9), even against opposing loads as high as ϳ6 pN (10 -12). The steps are driven by the hydrolysis of ATP; kinesin is an ATPase (13) whose speed of movement increases linearly with ATP concentration until it approaches a maximum of about 800 nm/s (8, 14), and AMP-PNP, 1 a nonhydrolyzable analog of ATP, arrests movement (15, 16). Despite intensive biochemical, biophysical, and structural investigations over the last few years, there remains considerable uncertainty as to the mechanism by which the stepping is coupled to ATP hydrolysis.In this work, we address the question: what is the stoichiometry of kinesin? In other words, how many steps does kinesin take for each ATP that it hydrolyzes? This question is important because it tests different models proposed to explain how motor proteins such as kinesin and myosin work. For example, one class of "thermal ratchet" models predicts that the stoichiometry is Յ0.5 steps/ATP (17, 18); these models postulate that ATP hydrolysis rectifies a diffusive motion in such a way that a step only occurs if the diffusion is toward the next binding site. Because there is an equal probability that the motor diffuses away from the next binding site (i.e. in the wrong direction), this model predicts that on average at least two molecules of ATP are hydrolyzed per forward step. A stoichiometry of less than one could also be due to "futile" hydrolysis cycles, those that fail to produce steps and lead to "slippage" between the mechanical and chemical cycles. Other models postulate a stoichiometry greater than 1 and have been...

Elastic Coupling Effects in Cooperative Transport by a Pair of Molecular Motors

“…1). Since we are interested in the movement of the cargo on length scales that are large compared to the motor step size l ' 8 nm, 17 we characterize each cargo state with an (average) velocity (v 1 and v 2 ). Transitions between the cargo states occur when a motor binds to or unbinds from the filament.…”

“…Most importantly they may be able to generate larger overall forces. 60 In addition motor cooperation can lead to longer runs, 3,10,17 in extreme cases up to millimeter. 12 Motor cooperation may also result in an increased velocity in situations where the motors work against an opposing load force if multiple motors share the load.…”

Microtubule transport, concentration and alignment in enclosed microfluidic channels

“…In some experiments, headless kinesin was used, which is a modified kinesin construct that contains the rod and tail domains and a hexaHis tag, but lacks its motor domain (Hancock and Howard, 1998). All motors were expressed in bacteria and purified by Ni column chromatography as previously described (Hancock and Howard, 1998;Coy et al, 1999). Tubulin was purified from bovine brains and labeled with rhodamine as previously described (Williams and Lee, 1982;Hyman et al, 1991).…”

Kinesin motors as molecular machines

“…(29) ATP hydrolyzed. (33,34) Thermal fluctuations are expected to play an important role in the motor mechanism, either by driving diffusional movements of the motor to its next binding position (35) or by promoting diffusive structural changes that drive force-producing conformational changes. (30) The structural elements that undergo strain have not been identified with certainty for any molecular motor, but are likely to have spring-like or elastic properties that allow them to extend or rotate, then recoil back into their original conformation.…”