Coupling of kinesin steps to ATP hydrolysis

Hua, Wei; Young, Edgar C.; Fleming, Margaret; Gelles, Jeff

doi:10.1038/41118

Cited by 321 publications

(230 citation statements)

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“…Here we consider conventional kinesin, ('kinesin', the Kinesin-1 subfamily) and Eg5 (the Kinesin-5 subfamily), both of which have conserved N-terminal catalytic motor domains and walk processively toward the plus-end of microtubules, hydrolyzing one ATP per 8-nm step ( [2,3,4••], reviewed in [5•,6,7,8•,9]). …”

Section: Introductionmentioning

confidence: 99%

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

Valentine

Gilbert

2007

Current Opinion in Cell Biology

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Conventional kinesin and Eg5 are essential nanoscale motor proteins. Single-molecule and presteady-state kinetic experiments indicate that both motors use similar strategies to generate movement along microtubules, despite having distinctly different in vivo functions. Single molecules of kinesin, a long-distance cargo transporter, are highly processive, binding the microtubule and taking 100 or more sequential steps at velocities of up to 700 nm/s before dissociating, whereas Eg5, a motor active in mitotic spindle assembly, is also processive, but takes fewer steps at a slower rate. By dissecting the structural, biochemical and mechanical features of these proteins, we hope to learn how kinesin and Eg5 are optimized for their specific biological tasks, while gaining insight into how biochemical energy is converted into mechanical work.

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Section: Introductionmentioning

confidence: 99%

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

Valentine

Gilbert

2007

Current Opinion in Cell Biology

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“…Tight coupling, i.e. an 8 nm step for every hydrolyzed ATP and a hydrolyzed ATP for every 8 nm step, has been observed for kinesin [8,9]. Without a mechanical load it is just the G ATP that is driving the cycle in Fig.…”

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confidence: 99%

How occasional backstepping can speed up a processive motor protein

Bier¹,

Cao²

2011

Biosystems

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Fueled by the hydrolysis of ATP, the motor protein kinesin literally walks on two legs along the biopolymer microtubule. The number of accidental backsteps that kinesin takes appears to be much larger than what one would expect given the amount of free energy that ATP hydrolysis makes available. This is puzzling as more than a billion years of natural selection should have optimized the motor protein for its speed and efficiency. But more backstepping allows for the production of more entropy. Such entropy production will make free energy available. With this additional free energy, the catalytic cycle of the kinesin can be speeded up. We show how measured backstep percentages represent an optimum at which maximal net forward speed is achieved. Processive motor proteins are among the tiniest engines known to man. These proteins utilize the energy of ATP hydrolysis to literally walk along a biopolymer [1]. In a living cell they help maintain organization by transporting cargo, like organelles or vesicles filled with chemicals.Already one and a half decade ago the stepping of the processive motor protein kinesin was made visible on the nanometer scale with optical tweezers [1]. Early communications [2,3] reported that 5% to 10% of all steps of kinesin were backward. But smaller fractions were described later on as methods and materials improved and better resolutions were achieved; Ref.[4] gave 1/220 and Ref.[5] gave 1/802. Theoreticians have always been interested in backstep fractions as they can help verify stochastic models. However, throughout the literature backstepping has implicitly and explicitly been seen as an occasional malfunction of the stepping motor protein. In this Letter we will show how, in the Brownian environment of the motor protein, a "well-tuned" backstep fraction can actually help the motor speed up. We will show how the backstep fraction that leads to the highest net speed can be evaluated and how the resulting expression contains no freely adjustable parameters. Finally, we will see how the experimentally established backstep fraction of kinesin is close to our predicted optimal backstep fraction.The operation of an ion pump is generally modeled with a cycle as depicted in Fig. 1. At equilibrium the product of the forward rates, k 12 × k 23 × ... × k n1 , equals the product of the backward rates, k 21 × k 32 × ... × k 1n , and no net cycling occurs. To drive the protein through the sequence of states, S 1 , S 2 ... S n , a driving energy is necessary [6]. Such energy comes available if one of the steps involves the binding of ATP and if the protein, in subsequent steps, catalyzes the hydrolysis of the bound ATP. Eventually the remaining ADP and an inorganic phosphate have to be released so as to complete the cycle and to put the protein again in a state in which it can bind a new ATP. Under physiological conditions the hydrolysis of ATP makes G ATP = 22 k B T units of free energy available. In the course of a cycle of a membrane pump like Na,K-ATPase, part of G ATP is utilized to bind, t...

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“…Single molecule motility studies have shown that kinesin can move along a single protofilament (6) in steps of 8 nm (the size of the tubulin dimer) each time that it hydrolyzes an ATP (7,8). Unlike many other molecular motors, such as muscle myosin, conventional kinesin is a highly processive motor and can take >100 steps along a microtubule before dissociating from the filament (9).…”

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ATPase Kinetic Characterization and Single Molecule Behavior of Mutant Human Kinesin Motors Defective in Microtubule-Based Motility

et al. 2000

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Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar ∼3-fold reduction in both microtubule gliding velocity and microtubulestimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.

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Coupling of kinesin steps to ATP hydrolysis

Cited by 321 publications

References 26 publications

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

How occasional backstepping can speed up a processive motor protein

ATPase Kinetic Characterization and Single Molecule Behavior of Mutant Human Kinesin Motors Defective in Microtubule-Based Motility

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