A universal pathway for kinesin stepping

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Unbranched cycles have been proposed to explain weaker ATP dependence of WT step ratio, which in this model mainly follows {1}->{2}->{5}->{1}.

“Unbranched cycles, which lack this property, have been proposed to explain the much weaker ATP dependence of the step ratio for WT kinesin22,23, which, in the context of this model, predominantly follows the sequence {1}→{2}→{5}→{1}.”

• cites [22] Carter, N.J. & Cross, R.A. Mechanics of the kinesin step. Nature 435, 308–312 (2005). sourceNot checked — no full text of the source
• cites [23] Hyeon, C., Klumpp, S. & Onuchic, J.N. Kinesin′s backsteps under mechanical load. Phys. Chem. Chem. Phys. 11, 4899–4910 (2009). sourceDisputed

  The paper makes the contrary point. Section V.A (p. 17) describes its explanation as a branched two-cycle model, and the Concluding Remarks (p. 20) attribute the weak ATP dependence of the step ratio to cancellation between its forward and backward cycles. Section III.B (p. 12) says a single-cycle model would instead predict ATP dependence. The paper does not give the cited {1}→{2}→{5}→{1} sequence. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

May combine two claims.

Kinesin-1 moves toward the microtubule plus end in discrete ~8.2 nm steps by alternating its two heads in an asymmetric hand-over-hand manner.

“Kinesin-1 is a motor protein that proceeds unidirectionally towards the plus end of the microtubule in discrete, 8.2-nm steps1 by alternately advancing each of its two heads, walking in an asymmetric, hand-over-hand fashion2–4.”

• cites [1] Svoboda, K., Schmidt, C.F., Schnapp, B.J. & Block, S.M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993). sourceNot checked — no full text of the source
• cites [2] Asbury, C.L., Fehr, A.N. & Block, S.M. Kinesin moves by an asymmetric hand-over-hand mechanism. Science 302, 2130–2134 (2003). sourceSupported
• cites [3] Kaseda, K., Higuchi, H. & Hirose, K. Alternate fast and slow stepping of a heterodimeric kinesin molecule. Nat. Cell Biol. 5, 1079–1082 (2003). sourceNot checked — no full text of the source
• cites [4] Yildiz, A., Tomishige, M., Vale, R.D. & Selvin, P.R. Kinesin walks hand-over-hand. Science 303, 676–678 (2004). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Front head-gated models propose that tension lowers the front head's affinity for ATP until the rear head detaches.

“front head-gated models propose that tension reduces the binding affinity of the front head for ATP until the rear head has detached from the microtubule12,17,18.”

• cites [12] Rosenfeld, S.S., Fordyce, P.M., Jefferson, G.M., King, P.H. & Block, S.M. Stepping and stretching. How kinesin uses internal strain to walk processively. J. Biol. Chem. 278, 18550–18556 (2003). sourcePartly supported

  The Discussion (page unavailable) proposes that rearward strain blocks ATP binding to the leading head until the trailing head dissociates. This supports the proposed gating sequence, but the paper describes an effect on binding kinetics, not a measured reduction in ATP-binding affinity. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [17] Guydosh, N.R. & Block, S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain. Proc. Natl. Acad. Sci. USA 103, 8054–8059 (2006). sourceSupported
• cites [18] Klumpp, L.M., Hoenger, A. & Gilbert, S.P. Kinesin′s second step. Proc. Natl. Acad. Sci. USA 101, 3444–3449 (2004). sourceSupported

May combine two claims.

Inter-head tension is thought to develop through the neck-linker regions, with molecular dynamics simulations suggesting 15-35 pN.

“Substantial inter-head tension is consequently thought to be developed between the heads via the neck-linker regions: molecular dynamics simulations suggest that 15–35 pN of tension may be produced15,16.”

• cites [15] Hyeon, C. & Onuchic, J.N. Internal strain regulates the nucleotide binding site of the kinesin leading head. Proc. Natl. Acad. Sci. USA 104, 2175–2180 (2007). sourcePartly supported

  The Results and Discussion describes tension transmitted through the neck-linker, supporting that part of the claim. But its estimate is 12–15 pN, calculated using a worm-like chain model of simulated structures, not the attributed 15–35 pN range. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [16] Hariharan, V. & Hancock, W.O. Insights into the mechanical properties of the kinesin neck-linker domain from sequence analysis and molecular dynamics simulations. Cell Mol. Bioengineering 2, 177–189 (2009). sourceNot checked — no full text of the source

May combine two claims.

At low ATP, WT kinesin is predominantly in State {1}, with leading head nucleotide-free and strongly bound, trailing head carrying ADP detached or transiently weakly associated.

“At low ATP concentrations, WT kinesin is found predominantly in this state, where the leading head is without nucleotide and strongly bound to the microtubule, while the trailing head, carrying ADP, remains detached from the microtubule34, or is perhaps transiently (weakly) associated36,37.”

• cites [34] Guydosh, N.R. & Block, S.M. Direct observation of the binding state of the kinesin head to the microtubule. Nature 461, 125–128 (2009). sourceSupported
• cites [36] Mori, T., Vale, R.D. & Tomishige, M. How kinesin waits between steps. Nature 450, 750–754 (2007). sourceNot checked — no full text of the source
• cites [37] Toprak, E., Yildiz, A., Hoffman, M.T., Rosenfeld, S.S. & Selvin, P.R. Why kinesin is so processive. Proc. Natl. Acad. Sci. USA 106, 12717–12722 (2009). sourceSupported

Classified as probably not a substantial claim.

Mechanical strain inside the kinesin molecule is thought to underlie how kinesin gates its heads' enzymatic activity.

“Mechanical strain developed within the kinesin molecule is thought to underlie the mechanism by which kinesin gates the enzymatic activity of its heads11–13.”

• cites [11] Guydosh, N.R. & Block, S.M. Not so lame after all: kinesin still walks with a hobbled head. J. Gen. Physiol. 130, 441–444 (2007). sourcePartly supported

  The main text says the heads’ catalytic cycles are mutually gated and describes evidence that the front head binds or hydrolyzes ATP more slowly than the rear. It does not explicitly say that strain within kinesin causes this gating; strain appears in the titles of cited works in the References section (page unavailable). (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [12] Rosenfeld, S.S., Fordyce, P.M., Jefferson, G.M., King, P.H. & Block, S.M. Stepping and stretching. How kinesin uses internal strain to walk processively. J. Biol. Chem. 278, 18550–18556 (2003). sourceSupported
• cites [13] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

In rear head-gated models, force from the leading head speeds release of the trailing head.

“Rear head-gated models propose that tension from the front head accelerates the detachment of the rear head13,19,20.”

• cites [13] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008). sourceSupported
• cites [19] Crevel, I.M. et al. What kinesin does at roadblocks: the coordination mechanism for molecular walking. EMBO J. 23, 23–32 (2004). sourceSupported
• cites [20] Schief, W.R., Clark, R.H., Crevenna, A.H. & Howard, J. Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism. Proc. Natl. Acad. Sci. USA 101, 1183–1188 (2004). sourceNot checked — no full text of the source

May combine two claims.

Similar unbranched forward-step pathways have been proposed, and an analogous 3-state cycle was used to model single-molecule data, but these do not account for backstepping and futile hydrolysis.

“Similar, unbranched reaction pathways for forward steps have been proposed38 based on the available mechanochemical, biochemical, and structural data, and an analogous 3-state cycle was used to model single-molecule data32. However, these pathways do not account for backstepping and futile hydrolysis.”

• cites [38] Valentine, M.T. & Gilbert, S.P. To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5. Curr. Opin. Cell Biol. 19, 75–81 (2007). sourceSupported
• cites [32] Valentine, M.T., Fordyce, P.M., Krzysiak, T.C., Gilbert, S.P. & Block, S.M. Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nat. Cell Biol. 8, 470–476 (2006). sourceNot checked — no full text of the source

May combine two claims.

The slower phase has kinetics consistent with motor dissociation from microtubule, as it is absent in low ionic-strength buffer.

“The slower phase has kinetics consistent with the subsequent dissociation of the motor from the microtubule, because this phase is absent when the experiment is repeated in low ionic-strength buffer12.”

• cites [12] Rosenfeld, S.S., Fordyce, P.M., Jefferson, G.M., King, P.H. & Block, S.M. Stepping and stretching. How kinesin uses internal strain to walk processively. J. Biol. Chem. 278, 18550–18556 (2003). sourceCould not verify

  The passage on two-phase 2′dmT fluorescence (section labeled Abstract; page unavailable) proposes that its second phase reflects nucleotide binding to the leading head, not motor dissociation. A separate passage reports nearly identical dissociation results at 10 mM KCl, but does not describe a slower phase disappearing in low ionic-strength buffer. Searches for that condition and phase did not locate the claimed observation in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Kinesin dimers labeled with fluorescent ADP analog release one ADP upon microtubule binding, and release of second requires ATP.

“It has long been known39 that kinesin dimers labeled with a fluorescent ADP analog will release just one ADP molecule upon binding the microtubule, while release of the second ADP requires the further addition of ATP39,40.”

• cites [39] Hackney, D.D. Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. Proc. Natl. Acad. Sci. USA 91, 6865–6869 (1994). sourceNot checked — no full text of the source
• cites [40] Alonso, M.C. et al. An ATP gate controls tubulin binding by the tethered head of kinesin-1. Science 316, 120–123 (2007). sourcePartly supported

  The passage discussing Fig. 3 reports that fluorescent mantADP is released from one kinesin head on binding unpolymerised tubulin, with release from the second head requiring an ATP or AMPPNP chase. It also describes the same two-stage ADP-release pattern on microtubule binding, but does not say that the microtubule result used fluorescently labeled ADP. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

Kin6AA remains well gated despite its extended neck-linker, which indicates that gating depends on more than inter-head tension.

“Although an extended neck-linker is thought to mitigate the effectiveness of strain-based gating mechanisms13,41, our results clearly indicate that Kin6AA is still well gated, and therefore support the notion that there is more to gating than the tension exerted between heads13,34.”

• cites [13] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008). sourceSupported
• cites [41] Hackney, D.D., Stock, M.F., Moore, J. & Patterson, R.A. Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups. Biochemistry 42, 12011–12018 (2003). sourceNot checked — no full text of the source
• cites [34] Guydosh, N.R. & Block, S.M. Direct observation of the binding state of the kinesin head to the microtubule. Nature 461, 125–128 (2009). sourceSupported

May combine two claims.

This intermediate signal is consistent with one head strongly bound while partner tethered, or with a mixture of singly- and doubly-bound head states.

“This finding is consistent with a single head being strongly bound to the microtubule while its partner remains tethered, or alternatively, with an admixture of singly- and doubly-bound head states43,44.”

• cites [43] Kawaguchi, K. & Ishiwata, S. Nucleotide-dependent single- to double-headed binding of kinesin. Science 291, 667–669 (2001). sourceNot checked — no full text of the source
• cites [44] Gutiérrez-Medina, B., Fehr, A.N. & Block, S.M. Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping. Proc. Natl. Acad. Sci. USA 106, 17007–17012 (2009). sourceSupported

May combine two claims.

In the ATP-waiting state, the trailing ADP-bearing head is mobile and detached from the microtubule, so it cannot put substantial tension on the leading head.

“It is also supported by results showing that in the ATP-waiting state, the trailing head carrying ADP is mobile and free of the microtubule34, and therefore unable to exert substantial tension on the leading head.”

• cites [34] Guydosh, N.R. & Block, S.M. Direct observation of the binding state of the kinesin head to the microtubule. Nature 461, 125–128 (2009). sourceSupported

May combine two claims.

The initial rise reflects neck-linker separation, with kinetics consistent with ATP-induced forward stepping.

“The initial increase reflects the separation of the neck-linkers, and its kinetics are consistent with ATP-induced forward stepping12,37.”

• cites [12] Rosenfeld, S.S., Fordyce, P.M., Jefferson, G.M., King, P.H. & Block, S.M. Stepping and stretching. How kinesin uses internal strain to walk processively. J. Biol. Chem. 278, 18550–18556 (2003). sourcePartly supported

  The paper describes ATP-triggered forward stepping and neck-linker separation, and its Discussion describes forward stepping as very rapid (about 800 s⁻¹). It does not identify the citing document’s initial increase as a signal of neck-linker separation; the fluorescence rise described in its own Results is attributed to nucleotide binding. No page numbers are available in our copy. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [37] Toprak, E., Yildiz, A., Hoffman, M.T., Rosenfeld, S.S. & Selvin, P.R. Why kinesin is so processive. Proc. Natl. Acad. Sci. USA 106, 12717–12722 (2009). sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Those neck-linker mutants moved uniformly slower than wild-type kinesin, but assisting load substantially raised their velocities.

“It was discovered that the neck-linker mutants moved uniformly slower along microtubules than wild-type kinesin, but that their velocities could be increased substantially via the application of assisting load.”

• cites [13] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008). sourceSupported

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.

It was conjectured that extra inter-head tension from ATP-induced neck-linker docking raised the rear-head detachment rate and largely coordinated the heads.

“Based on this property, it was conjectured that additional inter-head tension, due to ATP-induced neck-linker docking, increased the rate of rear-head detachment and was largely responsible for coordination between the heads.”

• cites [13] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008). sourceSupported

May combine two claims.

Non-hydrolyzable ATP analogs such as AMP-PNP or ADP-BeFx stop kinesin motion, and an obligatory backstep is needed to release the analog before ATP-driven forward motion can resume.

“When kinesin binds a non-hydrolyzable ATP analog, such as AMP–PNP or ADP–BeFx, its motion becomes arrested, and an obligatory backstep becomes necessary to unbind the analog before allowing any forward motion to continue in the presence of ATP17.”

• cites [17] Guydosh, N.R. & Block, S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain. Proc. Natl. Acad. Sci. USA 103, 8054–8059 (2006). sourcePartly supported

  The Results report that AMP-PNP or BeFx induced long pauses that almost invariably ended with a terminal backstep before forward motion resumed. The paper describes release of the bound analog after that backstep as a proposed mechanism—“presumably” allowing release in the opening passage and as part of a model in the Discussion—not as an established necessity. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

The analog can be released only from the leading head, not the trailing head, so a backstep is required to exchange head positions whenever the former trailing head bound the analog.

“It was concluded that the analog could only be released from the leading head, and not the trailing one, necessitating a backstep to interchange head positions whenever the (former) trailing head bound analog.”

• cites [17] Guydosh, N.R. & Block, S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain. Proc. Natl. Acad. Sci. USA 103, 8054–8059 (2006). sourcePartly supported

  The Discussion (page unavailable) says that processive stepping resumes only after an obligatory backstep exchanges the heads, permitting analog release from the new front head. But the paper describes release as preferential and the front head’s analog affinity as lower than the rear head’s; it does not establish that release from the rear head is impossible. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Yildiz et al. studied kinesin mutants carrying 2-26 extra prolines in the neck-linkers, including one with six extra prolines, using single-molecule optical trapping.

“A previous study of kinesin mutants with a series of neck-linker extensions was carried out by Yildiz et al.13. In that work, 2–26 additional prolines were inserted into the neck-linkers of kinesin, including one mutant with an extra six prolines, and the behavior was characterized by single-molecule, optical trapping assays.”

• cites [13] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008). sourcePartly supported

  The ‘Engineering Kinesin Motors with “Extended” Neck Linkers’ section lists 2P through 26P constructs, including 6P. The ‘Optical Trapping Assays’ section specifies optical-trap measurements for WT, 13P, 19P, and 14GS—not 6P or the full proline series. Thus the claim is supported for the mutants and for use of optical trapping, but overstates which mutants were characterized that way. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

For Kin6AA, mean velocity zero and forward/backward step ratio unity occurred at the same force for each ATP concentration.

“These metrics were mutually consistent (Fig. 2a,b), with v = 0 and SR = 1 occurring at the identical force for each ATP concentration.”

The quote only partly states this claim.

Kin6P, a construct with a 9-residue insert containing six adjacent prolines in its linker, gave essentially identical results to Kin6AA.

“Essentially identical results were obtained with a construct carrying a 9-residue insert with 6 adjacent prolines in its linker (Kin6P).”

May combine two claims.

The probability of a step in any direction is uncorrelated with direction of preceding step, confirming Markov property.

“the probability of a step in any direction is uncorrelated with the direction of the preceding step (Supplementary Fig. 1).”

May combine two claims.

Unlike WT kinesin, the mutant constructs' stall force varied greatly with ATP concentration, and average velocity smoothly reversed to negative as hindering load increased beyond stall.

“In contrast to wild-type (WT) kinesin, the stall force of the mutant constructs varied greatly with the ATP concentration, and the average velocity smoothly reversed and became negative as the hindering load was increased beyond stall.”

May combine two claims.

Kin6AA's average backward step size was 8.12 nm, indicating it uses the same microtubule binding sites as forward stepping.

“The backward step size for Kin6AA averaged 8.12 nm, indicating that the motor accesses the same microtubule binding sites as during forward stepping”

May combine two claims.

Kin6AA near stall remained bound and took many forward and backward steps with equal probability, producing large mean-squared displacement but no net progress.

“By contrast, Kin6AA motors approaching stall remained bound to the microtubule while undertaking numerous forward and backward steps with equal probability, leading to a large mean-squared displacement but no net progress”

May combine two claims.

Beyond stall, Kin6AA stepped rearwards processively with slow velocity strongly dependent on ATP concentration but relatively insensitive to load.

“As the force was increased beyond stall, Kin6AA began to step rearwards processively, with a slow velocity that was strongly dependent upon the ATP concentration, but comparatively insensitive to load”

May combine two claims.

When TMR-labeled KinWT was mixed with ATP, fluorescence rose rapidly then fell slowly.

“When this procedure was performed using KinWT, a rapid initial rise in fluorescence was observed, followed by a slower exponential fall”

May combine two claims.

A short lag follows the initial rise, then a slower exponential fluorescence increase occurs with a rate constant near 3 s−1 and little dependence on 2′dmT concentration.

“Next came a brief lag phase, followed in turn by a second, slower exponential increase with a rate constant of ~3 s−1 that displayed little 2′dmT concentration-dependence (Fig. 5b, inset).”

• AssumptionRests on an assumption: The FRET signal between tryptophan residues and the mant fluorophore in 2′dmT reports 2′dmT binding to kinesin.

May combine two claims.

Without microtubules, Kin6AA mixed with 2′dmT gives a fast single-phase fluorescence rise whose amplitude is similar to that seen with microtubules.

“This interpretation is bolstered by the observation that mixing Kin6AA with 2′dmT in the absence of microtubules produces a rapid increase in fluorescence consisting of a solitary phase, with amplitude similar to that in the presence of microtubules (Fig. 5a, grey).”

• AssumptionRests on an assumption: The FRET signal between tryptophan residues and the mant fluorophore in 2′dmT reports 2′dmT binding to kinesin.

May combine two claims.

The decay has two phases; faster phase is 80-85% of amplitude and has kinetics consistent with dissociation of new trailing head to regenerate a singly-attached species.

“The faster phase constitutes 80–85% of the total amplitude of the decay phase, and it has kinetics consistent with the dissociation of the new trailing head from the microtubule to regenerate a singly-attached species.”

May combine two claims.

The initial fast fluorescence rise follows a single exponential, and its rate constant varies hyperbolically with 2′dmT concentration.

“A rapid initial increase could be fit to a single exponential process, with a rate constant that displayed a hyperbolic dependence on 2′dmT concentration (Fig. 5b).”

• AssumptionRests on an assumption: The FRET signal between tryptophan residues and the mant fluorophore in 2′dmT reports 2′dmT binding to kinesin.

May combine two claims.

After ~2 ms delay, Kin6AA fluorescence quenched at an initial rate dependent on ATP concentration.

“After a delay of ~2 ms, fluorescence was quenched at an initial rate that depended upon ATP concentration.”

May combine two claims.

Fitting the concentration dependence of this decay to a hyperbola gave a y-intercept of 11 ± 4 s−1, interpreted as trailing head rebinding rate.

“Fitting the concentration dependence of this decay to a hyperbola generates a curve with a y-intercept of 11 ± 4 s−1 (Fig. 5), which we interpret as the rate at which the trailing head rebinds the microtubule.”

May combine two claims.

TMR-labeled KinWT produced a large fluorescence signal with AMP-PNP and microtubules, indicating neck-linkers separate when motor binds microtubule.

“TMR-labeled KinWT produced a large fluorescence signal when incubated with AMP–PNP and microtubules (Fig. 4a), indicating that its neck-linkers separate when the motor binds the microtubule.”

May combine two claims.

Kin6AA is more processive than KinWT, with runs averaging more than 150 steps.

“Indeed, the processivity of Kin6AA exceeds that of the KinWT construct, supporting long runs that average over 150 steps (Supplementary Fig. 5).”

May combine two claims.

Kin6AA shows more backstepping under load and clear decreases in speed, stall force, and energy efficiency.

“However, a price is clearly paid, through increased backstepping under load and marked reductions in both speed and stall force, along with energy efficiency.”

May combine two claims.

The three transition rates involving forward step (k23) or rear-head rebinding during backstep (k14, k25) carry load dependence and were modeled by Boltzmann expressions.

“The three transition rates that involve taking a forward step (k23) or rebinding the rear head to the microtubule during a backstep (k14, k25) were found to carry load dependence an”

May combine two claims.

Kin6AA permits neck-linker separation on microtubules both with AMP-PNP and without nucleotide.

“By contrast, Kin6AA not only permits neck-linker separation on microtubules in the presence of AMP–PNP, but also in the absence of nucleotide”

May combine two claims.

The model fits force-velocity data, randomness, and stepping ratios versus load, and reproduces the apparent Michaelis-Menten dependence of velocity on ATP concentration.

“It fits well to the experimentally determined force-velocity data, as well as to the randomness and the stepping ratios as functions of load, while capturing the apparent Michaelis-Menten behavior of velocity as a function of ATP concentration.”

May combine two claims.

Because r−1 is proportional to mean velocity, randomness changes sign and crosses zero at stall.

“Because r−1 is proportional to the mean velocity, the randomness value changes sign and crosses the abscissa at stall.”

May combine two claims.

Strong binding of both heads to the microtubule places them 8.15 nm apart, matching the tubulin-dimer lattice spacing.

“When both heads bind strongly to the microtubule, they become separated by a distance corresponding to the tubulin-dimer lattice spacing of 8.15 nm.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Prior work showed that ATP binding, but not necessarily hydrolysis, is required for backstepping.

“Investigations into the prevalence of backward steps near the stall force21 and beyond22 have demonstrated that ATP binding, but not necessarily hydrolysis, is required to backstep.”

• cites [21] Nishiyama, M., Higuchi, H. & Yanagida, T. Chemomechanical coupling of the forward and backward steps of single kinesin molecules. Nat. Cell Biol. 4, 790–797 (2002). sourceNot checked — no full text of the source
• cites [22] Carter, N.J. & Cross, R.A. Mechanics of the kinesin step. Nature 435, 308–312 (2005). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Kinesin motion is highly processive, with single motors usually taking about 100 steps before detaching.

“Kinesin motion is remarkably processive, with single motors typically taking ~100 steps before detaching from the microtubule5.”

• cites [5] Block, S.M., Goldstein, L.S. & Schnapp, B.J. Bead movement by single kinesin molecules studied with optical tweezers. Nature 348, 348–352 (1990). sourceNot checked — no full text of the source

May combine two claims.

Backsteps toward the minus end are rare and appear mainly when external loads are near or above stall.

“Deviations from that cycle, such as those resulting in backsteps towards the microtubule minus end, occur only rarely, or in response to external loads that approach or exceed the stall force9.”

• cites [9] Svoboda, K. & Block, S.M. Force and velocity measured for single kinesin molecules. Cell 77, 773–784 (1994). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

The reciprocal randomness parameter r−1 has been used to distinguish competing kinetic pathways for kinesin.

“The reciprocal randomness, r−1, has been used to distinguish between competing kinetic pathways for kinesin8,32.”

• cites [8] Schnitzer, M.J. & Block, S.M. Kinesin hydrolyses one ATP per 8-nm step. Nature 388, 386–390 (1997). sourceNot checked — no full text of the source
• cites [32] Valentine, M.T., Fordyce, P.M., Krzysiak, T.C., Gilbert, S.P. & Block, S.M. Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nat. Cell Biol. 8, 470–476 (2006). sourceNot checked — no full text of the source

May combine two claims.

The front- and rear-head gating models are not mutually exclusive, and kinesin may use both.

“These models are not mutually exclusive, and kinesin may utilize both mechanisms to maintain coordination during processive stepping.”

Classified as probably not a substantial claim. May combine two claims.

The neck-linker is a short, largely unstructured ~14 amino acid sequence connecting each kinesin head to the common stalk, and it has been implicated in the conformational change thought to drive motility.

“The neck-linker consists of a short, largely unstructured ~14 amino acid sequence that connects each of the kinesin heads to the common stalk, and it has been implicated in the conformational change thought to drive motility25.”

• cites [25] Rice, S. et al. A structural change in the kinesin motor protein that drives motility. Nature 402, 778–784 (1999). sourceNot checked — no full text of the source

May combine two claims.

A successful backstep requires the trailing head to bind microtubule, forming a two-heads-bound state and releasing ADP from rear head.

“First, the trailing head must be able to bind the microtubule to generate a two-heads-bound state, leading to release of ADP from the rear head.”

May combine two claims.

In the model, reversible ATP binding can occur either before rear head binds microtubule ({1}->{2}->{5}->{1}) or after ({1}->{4}->{5}->{1}).

“In the model, reversible ATP binding can occur in two ways: either prior to the rear head binding the microtubule, following the sequence {1}→{2}→{5}→{1}, or afterwards, following the sequence {1}→{4}→{5}→{1}.”

Classified as probably not a substantial claim.

Because Kin6AA has an extended linker designed to relieve inter-head tension, its trailing head may transiently bind/unbind microtubule from State {1} if ADP release is not too facile.

“Because Kin6AA carries an extended linker designed to relieve inter-head tension, it is possible, in principle, for its trailing head to bind and unbind the microtubule transiently from this state, provided that this conformation is not excessively prone to releasing its bound ADP.”

• AssumptionRests on an assumption: The extended neck-linker in Kin6AA reduces inter-head tension.
• AssumptionThe authors state this as an assumption: In Kin6AA, the trailing head can transiently bind and unbind the microtubule from State {1} without releasing its bound ADP too readily.

May combine two claims.

Dwell-time distributions can differ greatly among kinetic models with the same cycle time, so a metric like randomness is needed to distinguish them.

“In practice, distributions of dwell times can vary enormously for different kinetic models with otherwise identical cycle times, requiring a metric such as randomness to differentiate among them33.”

• cites [33] Moffitt, J.R., Chemla, Y.R. & Bustamante, C. Methods in statistical kinetics. Methods Enzymol. 475, 221–257 (2010). sourceNot checked — no full text of the source

May combine two claims.

Adding a state where both heads bind ATP and remain bound would introduce additional futile hydrolysis and backstepping pathways, increasing complexity without new observable features.

“The existence of such a state would introduce one additional pathway for futile hydrolysis as well as one for backstepping, increasing the model complexity but without adding any new observable features.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Simultaneous release of both nucleotides supporting a two-heads-bound state was previously reported by Hackney and coworkers for a different extended-neck-linker mutant.

“The simultaneous release of both nucleotides, with consequent support for a two-heads-bound state, was previously reported by Hackney and coworkers for a different mutant construct that also carried an extended neck-linker.41”

• cites [41] Hackney, D.D., Stock, M.F., Moore, J. & Patterson, R.A. Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups. Biochemistry 42, 12011–12018 (2003). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Results from model fits supply predictions about molecular configurations and transitions.

“Results from fits to the model in Figure 3 supply predictions about molecular configurations and transitions between states.”

May combine two claims.

Futile hydrolysis is suppressed in WT, giving greater efficiency and about 1 ATP per forward step.

“Futile hydrolysis is suppressed in the WT, leading to greater efficiency and ~1 ATP per forward step6–8.”

• cites [6] Coy, D.L., Wagenbach, M. & Howard, J. Kinesin takes one 8-nm step for each ATP that it hydrolyzes. J. Biol. Chem. 274, 3667–3671 (1999). sourceNot checked — no full text of the source
• cites [7] Hua, W., Young, E.C., Fleming, M.L. & Gelles, J. Coupling of kinesin steps to ATP hydrolysis. Nature 388, 390–393 (1997). sourceNot checked — no full text of the source
• cites [8] Schnitzer, M.J. & Block, S.M. Kinesin hydrolyses one ATP per 8-nm step. Nature 388, 386–390 (1997). sourceNot checked — no full text of the source

May combine two claims.

In unloaded Kin6AA, reorientation of the head with the lengthened neck-linker is slow (~3 s−1), and this process, rather than relief of inter-head tension itself, gates ATP binding.

“In Kin6AA, the reorientation of the head with a lengthened neck-linker (in the absence of any external load) is a comparatively slow process, (~3 s−1, Fig. 5b inset) and it is this process, and not the relief of inter-head tension, per se, that gates ATP binding.”

May combine two claims.

Without futile hydrolysis, slow backstepping would cause extended dwell times and small r−1 at low forces, which were not observed.

“Without futile hydrolysis, the slow backstepping rate would result in extended dwell times, yielding small values of r−1 at low forces, which were not observed”

May combine two claims.

2′dmT can bind the second head only after the first head dissociates and the inter-head tension from microtubule binding is reduced.

“This result implies that binding of 2′dmT to the second head can only occur after dissociation of the first head, and the concomitant reduction of any inter-head tension imposed by microtubule binding.”

May combine two claims.

The unloaded forward-velocity versus ATP dataset for Kin6AA was excluded from model fitting, and the model's parameter-free prediction of that dataset matched the data excellently.

“In addition, the single-molecule dataset for the unloaded forward velocity of Kin6AA as a function of ATP was not used to fit the parameters of the model. The model should therefore predict this function independently, with no adjustable parameters: the agreement between the data and prediction was excellent (Supplementary Fig. 4).”

May combine two claims.

Moderate neck-linker lengthening in Kin6AA permits the trailing head to bind tightly to the microtubule and release ADP while still retaining substantial gating.

“The experiments with Kin6AA demonstrate that moderately lengthening the neck-linker allows the trailing head to bind strongly to the microtubule and release ADP, yet retain substantial aspects of gating.”

May combine two claims.

A forward docked neck-linker orientation is associated with fast ATP hydrolysis and slow ATP unbinding, while a rearward orientation is associated with slow hydrolysis and fast unbinding.

“The picture that emerges is that when the neck-linker is in its forward (docked) orientation, ATP hydrolysis is fast and ATP unbinding is slow. Conversely, when the neck-linker is in a rearward orientation, ATP hydrolysis is slow and ATP unbinding is fast.”

May combine two claims.

Trailing-head attachment is strain-based, and reduced inter-head tension makes this state more accessible.

“Our results do, however, support a mechanism whereby attachment of the trailing head is strain-based, with the accessibility of this state being promoted by a reduction in inter-head tension.”

May combine two claims.

Assisting load lowers the chance that the trailing head binds tightly to the microtubule because of the force dependence of the rebinding rates (k14, k25), raising average velocity and offering an alternative explanation for earlier results without invoking inter-head tension as a gating mechanism.

“The application of an assisting load decreases the probability that the trailing head will bind tightly to the microtubule, due to the force dependence of the rebinding rates (k14, k25), which effectively increases the average velocity: this supplies an alternate explanation for the earlier results, but without invoking inter-head tension as a gating mechanism.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The 2′dmD and 2′dmT products were purified to more than 98% purity.

“The 2′dmD and 2′dmT reaction products were purified by chromatography over a Sephadex LH20 column to >98% purity, determined from the relative absorbances at 255 and 356 nm, as described48.”

• cites [48] Hiratsuka, T. New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochim. Biophys. Acta 742, 496–508 (1983). sourceNot checked — no full text of the source

May combine two claims.

A gating mechanism based on neck-linker orientation can explain native kinesin's high processivity even under force, because it lowers the chance of ATP hydrolysis before a mechanical step is completed.

“A neck-linker orientation-based gating mechanism can explain the large processivity of native kinesin, even under force45, since it reduces the probability of ATP hydrolysis until a mechanical step has been completed.”

• cites [45] Schnitzer, M.J., Visscher, K. & Block, S.M. Force production by single kinesin motors. Nat. Cell Biol. 2, 718–723 (2000). sourceNot checked — no full text of the source

May combine two claims.

The only way out of a doubly-bound state is futile hydrolysis or a backstep, both of which reduce velocity and efficiency.

“The only escape from a doubly-bound state (Fig. 3b) is via futile hydrolysis or a backstep, both of which act to decrease the velocity and efficiency.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Data for forces above −3 pN were collected with force clamping using a feedback-based optical trap.

“All data for forces exceeding −3 pN were obtained under force clamped conditions using a feedback-based optical trapping system.”

May combine two claims.

The slower velocity of the neck-linker mutants is due to their greater tendency to enter a doubly-bound microtubule state, not to a large change in rear-head detachment rate, especially under hindering loads.

“Based on our results, we would interpret the decrease in velocity for the neck-linker mutants to be a consequence of their increased propensity to enter a doubly-bound state on the microtubule, rather than to a large modulation in the rear-head detachment rate, especially under hindering loads.”

May combine two claims.

The reaction rates obtained here suggest that kinesin head coordination is finely tuned and add insight into gating mechanisms.

“The values for the reaction rates obtained here suggest how exquisitely tuned head coordination is in kinesin, and shed additional light on the mechanisms responsible for gating.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For forces below −3 pN, data were collected without force feedback, causing a small force change during each step.

“However, for small forces below −3 pN, data were collected without force feedback, leading to a minor change in force during the course of each step.”

Classified as probably not a substantial claim.

The motor-to-bead ratio was set so that fewer than 25% of trapped kinesin-coated beads tested on microtubules showed motility.

“The motor protein-bead ratio was adjusted so that fewer than 25% of kinesin-coated beads captured by the optical trap and tested on microtubules showed motility.”

May combine two claims.

The model predicts high processivity because the mutants spend more of the cycle with both heads attached, lowering the chance of detachment from a one-head-bound state.

“High processivities are predicted by the model, since the mutants spend a greater portion of the reaction cycle with both heads attached to the microtubule, which reduces the chance of detachment from a one-head-bound state.”

May combine two claims.

Our results agree with front-head gating because the predicted nucleotide unbinding rate is almost six times faster in the leading head than in the trailing head.

“Our results are fully consistent with that interpretation, since the predicted rate of nucleotide unbinding is almost six-fold faster in the leading than the trailing head.”

Classified as probably not a substantial claim.

Preferential release of these analogs from the leading head was taken as evidence for front-head gating.

“Because such analogs are preferentially released from the leading head, these results were interpreted as evidence for front-head gating.”