Applied load and 40 mM phosphate act as checkpoints that control the flux of myosin V through two parallel kinetic pathways by altering forward and backward stepping probabilities.
Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (Pi), both forward and backward steps become less load-dependent. From these data, we infer that Pi release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of Pi. We propose a branched kinetic model to explain these data. Our model includes Pi release prior to the most load-dependent step in the cycle, implying that Pi release and load both act as checkpoints that control the flux through two parallel pathways. Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (Pi), both forward and backward steps become less load-dependent. From these data, we infer that Pi release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of Pi. We propose a branched kinetic model to explain these data. Our model includes Pi release prior to the most load-dependent step in the cycle, implying that Pi release and load both act as checkpoints that control the flux through two parallel pathways. Myosin V is a cargo-carrying molecular motor that converts chemical energy from ATP hydrolysis into 36-nm hand-over-hand strides, as it moves processively along actin tracks (1Reck-Peterson S.L. Provance Jr., D.W. Mooseker M.S. Mercer J.A. Biochim. Biophys. Acta. 2000; 1496: 36-51Crossref PubMed Scopus (243) Google Scholar). A striking feature of the molecule is its long α-helical neck domain, which binds six calmodulins in series (2Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (380) Google Scholar) and acts as a lever arm (3Sakamoto T. Wang F. Schmitz S. Xu Y. Xu Q. Molloy J.E. Veigel C. Sellers J.R. J. Biol. Chem. 2003; 278: 29201-29207Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 4Purcell T.J. Morris C. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14159-14164Crossref PubMed Scopus (146) Google Scholar, 5Moore J.R. Krementsova E.B. Trybus K.M. Warshaw D.M. J. Muscle Res. Cell Motil. 2004; 25: 29-35Crossref PubMed Scopus (41) Google Scholar, 6Schott D.H. Collins R.N. Bretscher A. J. Cell Biol. 2002; 156: 35-39Crossref PubMed Scopus (170) Google Scholar). Beyond the neck, the two heavy chains form a predominantly α-helical coiled-coil that ends in a globular cargo-binding domain, which is also involved in regulating the activity of the molecule (7Thirumurugan K. Sakamoto T. Hammer III, J.A. Sellers J.R. Knight P.J. Nature. 2006; 442: 212-215Crossref PubMed Scopus (140) Google Scholar, 8Liu J. Taylor D.W. Krementsova E.B. Trybus K.M. Taylor K.A. Nature. 2006; 442: 208-211Crossref PubMed Scopus (172) Google Scholar). For myosin V to travel processively over long, 1–2-μm distances, the ATPase activity and motion generation of the individual heads must be coordinated so that one head remains bound to actin as the other head steps forward (9Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Crossref PubMed Scopus (333) Google Scholar). This coordination requires the heads to communicate, presumably through intramolecular strain that develops as the leading head attempts to swing its lever arm forward but is resisted by the strongly bound trailing head (5Moore J.R. Krementsova E.B. Trybus K.M. Warshaw D.M. J. Muscle Res. Cell Motil. 2004; 25: 29-35Crossref PubMed Scopus (41) Google Scholar, 9Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Crossref PubMed Scopus (333) Google Scholar, 10De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar, 11Purcell T.J. Sweeney H.L. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13873-13878Crossref PubMed Scopus (147) Google Scholar). This internal resistive load may slow the release of ATP hydrolysis products (i.e. ADP and/or Pi) from the active site of the leading head, whereas the positive strain experienced by the trailing head may accelerate their release (10De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar, 11Purcell T.J. Sweeney H.L. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13873-13878Crossref PubMed Scopus (147) Google Scholar, 12Rosenfeld S.S. Sweeney H.L. J. Biol. Chem. 2004; 279: 40100-40111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Veigel C. Schmitz S. Wang F. Sellers J.R. Nat. Cell Biol. 2005; 7: 861-869Crossref PubMed Scopus (219) Google Scholar, 14Forgacs E. Cartwright S. Sakamoto T. Sellers J.R. Corrie J.E. Webb M.R. White H.D. J. Biol. Chem. 2008; 283: 766-773Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The specific biochemical and mechanical states that each head transitions through during its processive run is far from certain, but we and others have proposed that myosin V proceeds through a branched kinetic scheme (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar, 16Uemura S. Higuchi H. Olivares A.O. De La Cruz E.M. Ishiwata S. Nat. Struct. Mol. Biol. 2004; 11: 877-883Crossref PubMed Scopus (153) Google Scholar), potentially offering the myosin V molecule alternate processive pathways as it negotiates the crowded cytoskeletal network of the cell (17Luby-Phelps K. Castle P.E. Taylor D.L. Lanni F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4910-4913Crossref PubMed Scopus (333) Google Scholar) and the loads that this meshwork may present. To characterize the kinetic pathways of myosin V and the specific states that are sensitive to load, we have used the single molecule laser trap assay to examine the stepping kinetics of expressed double-headed myosin V heavy meromyosin in response to load. With increasing load, the attached lifetime following a forward step was significantly prolonged. At high forces, a dynamic equilibrium was reached where the probability of myosin V taking a forward or backward step was equal. Additional insight into the kinetic pathways taken by myosin V under load was obtained through changes in inorganic phosphate (Pi) concentration, because Pi release may be linked to the powerstroke and potentially in the presence of Pi J.A. J. E. J. PubMed Scopus Google Scholar, Y. H. Biol. Sci. 2004; PubMed Scopus Google Scholar). The presence of Pi significantly the load of the stepping kinetics of myosin the of load and Pi as that myosin V can multiple kinetic pathways and that each one or transitions are sensitive to load. and heavy meromyosin myosin and a protein was expressed in as (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar). This used by from expressed myosin heavy meromyosin D.M. Kennedy G.G. S.S. Krementsova E.B. S. Trybus K.M. Biophys. J. 2005; Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The is from the which is at a single during in Jr., J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Jr., J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was from as Spudich J.A. PubMed Scopus Google Scholar). The actin was used assay used mm mm mm mm active of Krementsova E.B. Trybus K.M. J. Cell Biol. 2004; PubMed Scopus Google and mm The was 40 mm phosphate prior to to or mm of the at and laser trap assay was the Kennedy G.G. Warshaw D.M. Trybus K.M. J. Cell Biol. 2003; PubMed Scopus Google Scholar). To the by at in as Kennedy G.G. Warshaw D.M. Trybus K.M. J. Cell Biol. 2003; PubMed Scopus Google Scholar, Y. Warshaw D.M. Trybus K.M. J. Muscle Res. Cell Motil. PubMed Scopus Google Scholar), myosin V was attached to the to its to the in the following of of 40 of myosin V and to a single myosin molecule the actin of assay of and of and a single was in each With actin in the was so that the ends of the actin attached to the the The actin was to at by the This was a that was to the and myosin We activity less of the that single The in this obtained from a of Pi and the Pi. molecule was of steps to be from We that trap to processive because of the that characterize this To the of the laser a was used J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In a was applied by the and laser trap Trybus K.M. Warshaw D.M. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The response is in of the obtained from the form of the trapped in Kennedy J. Warshaw D.M. Biophys. J. Full Text PDF PubMed Scopus Google the was by the response The was by a of the the of the laser trap its The of the laser was The of the to was each of myosin by stepping the and its the A processive run was it the of the or of the steps V stepping was by the of the that was from its trap The at and at transitions and by changes in a of The steps by and a lifetime by in the in the was used as the This of was to time and by a to to the as steps and and at loads but less at loads as because forward steps this a and At high to the prior to that the myosin V molecule can to actin This is the ATP hydrolysis (10De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar). At loads backward steps and in was used to steps that in a to in detachment of myosin V from as under the step of a was from its or forward and these as of the the to the in the or of load. The of the of steps was the at In the of Pi the load-dependent and to a single load-dependent and With Pi a in load is as as a Pi backstepping in the of load. A to and are because these are the from multiple single and in the presence and of 40 mm Pi. step forward the are to a the experienced by myosin V in the step detachment. The to a of as a but Pi and a of as but Pi and a of the of To the used by myosin V during a processive the attached of forward and backward of to forward and detachment kinetics under load in the presence and of Pi. myosin V steps the of the laser trap a and and the motor steps forward loads at trap the attached lifetime following a step that the stepping kinetics are The load of the forward step was by a PubMed Scopus Google Scholar) two load-dependent where the attached lifetime and are the the load-dependent at and are the to the transition is the is the and is in The to this and The of high of phosphate transitions the ATPase In the presence of 40 mm to that in the of myosin V processively multiple steps and and the prior to detachment at significantly loads that in the of phosphate The detachment was of laser trap that the detachment was by the run of the motor as under (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar). in the presence of phosphate processive at a and slow load as in the of Pi a and The load of the attached was by a single to the of a at load, and a the presence of Pi the that forward steps in the of Pi. and of be at loads and the attached following a load-dependent kinetics The by a single load-dependent to the of and are the highly load-dependent forward that these two may the The of backward to forward steps load At myosin V a dynamic where the probability of taking a step or is as by the to the detachment The of backward to forward steps equilibrium stepping that load and was to the following where is the is the equilibrium in the of is the applied is the in the equilibrium of the myosin V molecule as a of a is the and is in the the equilibrium of the motor by during a At load that one in steps be a the forward the presence of Pi a and highly attached lifetime following a to a and less load-dependent The load forward and steps in the presence of Pi was that the attached lifetime following a forward or step is by the transition in the processive The of to steps to in the of Pi. With the by of and indicate that the probability of a at load, but the of a is as sensitive to load as in the of Pi. in the of phosphate dynamic that the of detachment is in the presence of Pi. of the detachment kinetics of myosin V from actin can be from the attached of the step prior to detachment in the of Pi as which a H. J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar), and by a of and indicate that myosin V may detach from actin two In the presence of the attached of the step prior to of a processive run by a single a of that Pi one of the two detachment that in the of Pi. of the in the of of at loads by two and To the of these the of the forward steps also prior to a of two of and to a or forward of the in this we to where the of a is from the forward step each individual We two The was myosin V backstepping to its in both the of a and 36-nm forward The was and as a of a a 36-nm forward step and a myosin V steps forward a of The and 36-nm step may indicate that this step from a of steps a that is in the of the and of backsteps. Our that the of the motor to its actin as a of a forward step and This is the of this under load myosin V steps forward of the actin the of the molecule The lead head and the of the powerstroke of the head, steps to the actin the molecule to its The in is by a 36-nm forward step to two by a of of this the head steps to the actin in the lead head, in a forward step and a the actin Our that the forward stepping kinetics of myosin V are by two load-dependent S. Higuchi H. Olivares A.O. De La Cruz E.M. Ishiwata S. Nat. Struct. Mol. Biol. 2004; 11: 877-883Crossref PubMed Scopus (153) Google Scholar, Mooseker M.S. R.E. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, Spudich J.A. Mooseker M.S. R.E. Nature. 1999; PubMed Scopus Google Scholar). is and load-dependent the in attached under resistive loads The probability of increases load where the probability of taking a forward or a backward step is to of dynamic and Higuchi H. T. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, Nature. 2005; PubMed Scopus Google Scholar, A. S.L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), but myosin load slows myosin V and increases the probability of the motor as J. J. Biophys. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). a the load the attached lifetime is by the and forward stepping To insight into the of the load-dependent high of phosphate to through steps that involved phosphate The of the motor was by The most striking was that both forward steps and steps following a be by a single load to the forward steps in the of Pi We these to as as to a branched kinetic model of myosin V load-dependent kinetics forward stepping by a scheme of two transitions the ATPase of myosin these transitions Pi release from the leading head by ADP release from the trailing head this kinetic scheme proposed S.S. Sweeney H.L. J. Biol. Chem. 2004; 279: 40100-40111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Forgacs E. Cartwright S. Sakamoto T. Sellers J.R. Corrie J.E. Webb M.R. White H.D. J. Biol. Chem. 2008; 283: 766-773Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Mooseker M.S. R.E. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, A. Biophys. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), other branched kinetic to myosin V (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar, 16Uemura S. Higuchi H. Olivares A.O. De La Cruz E.M. Ishiwata S. Nat. Struct. Mol. Biol. 2004; 11: 877-883Crossref PubMed Scopus (153) Google Scholar). of (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar), the stepping kinetics in the that a branched kinetic model be we by the scheme in the of Pi have or at the of forward stepping at This was the that of Pi the motor along alternate that a single load-dependent transition With the load-dependent of at load to ADP release obtained both in and the laser trap (9Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Crossref PubMed Scopus (333) Google Scholar, 10De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar, 12Rosenfeld S.S. Sweeney H.L. J. Biol. Chem. 2004; 279: 40100-40111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), we propose that the alternate ADP from the trailing head state prior to Pi release from the leading head state In to the ADP release so is its to load as a myosin V C. Schmitz S. Wang F. Sellers J.R. Nat. Cell Biol. 2005; 7: 861-869Crossref PubMed Scopus (219) Google Scholar). is less the the double-headed of heavy meromyosin may the load of the trap to be the in a of the load experienced by the trailing head to A. Biophys. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the highly load-dependent transition in the presence of Pi also that the motor can travel along where state following Pi release must that is highly load-dependent This state have ADP bound to both the leading head in the pre-powerstroke conformation. a state and by (10De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar, 11Purcell T.J. Sweeney H.L. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13873-13878Crossref PubMed Scopus (147) Google Scholar, 12Rosenfeld S.S. Sweeney H.L. J. Biol. Chem. 2004; 279: 40100-40111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and potentially by S. Wang F. Sellers J.R. White H.D. Knight P.J. J. J. Cell Biol. 2002; PubMed Scopus Google Scholar). The transition of state requires the leading head to its lever arm the of the trailing head and the load, the high load We propose that this transition state to the of the motor M.L. Sellers J.R. Wang F. Hammer III, J.A. J. Knight P.J. Nature. 2000; PubMed Scopus Google Scholar). In flux through A is by the release of Pi from state the motor to the highly load-dependent state to state a transition in state it may be as load-dependent at the loads because of a in the internal strain the motor as a of the lever arm ADP release state C. Schmitz S. Wang F. Sellers J.R. Nat. Cell Biol. 2005; 7: 861-869Crossref PubMed Scopus (219) Google Scholar). ATP to the trailing head in state or release it from in the forward transition by internal strain or the run as both heads be in a This explain the motor dynamic in the presence of Pi and at J. J. Biophys. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) but (9Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Crossref PubMed Scopus (333) Google Scholar, Mooseker M.S. R.E. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). The may be to the in which the loads are or the myosin V both are of the attached the step prior to that myosin from actin by of two one at a slow of and a at the is in the presence of we it along to Pi run can at its probability is at high loads where the motor presumably in state detachment and run from this state at detachment proposed from a state a strongly bound head and a less bound lead head J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). of its to the detachment of must from a state to A and (i.e. state from state was proposed in model under (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar), where the of detachment and stepping forward the probability of run The that this increases to in the presence of Pi is by Pi the flux through where the is less load-dependent and under to at the loads the detachment in both the presence and of Pi the of the from state as as the motor can to this which is by the a branched kinetic a in A from a state that under high the motor in a strongly bound and kinetics of a branched kinetic as as insight into the states that the motor during its processive to be predominantly a at which we to be to a This in the biochemical cycle, which was by the of steps and following a We the forward step was a 36-nm was a and that this was of a single molecule to the of a and its forward This that myosin to its or of this The in the is by 36-nm from 36-nm forward and also by forward steps To explain the a from a 36-nm forward step a in the are the of myosin V backstepping a myosin V to its taking a or In the of a the lead head and actin of its the heads This step in the lead head to to the trailing The forward step the trailing head into the of the leading head by the actin this the moves and the is to This that under load, myosin V may forward steps and the actin S. K. H. Jr., K. Ishiwata S. Nat. Struct. Biol. 2002; PubMed Scopus Google Scholar). of the steps following a that the forward step is a of 36-nm and The in may cargo Krementsova E.B. Kennedy G.G. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). From which specific the The load attached following a is by a highly load-dependent to the of the two that the load of forward the motor steps it steps forward through the highly load-dependent step that forward steps transition through along A (i.e. state state the from state because the lifetime of steps a are from forward steps at the load both the step and kinetic that load, most a step and We propose that as the motor attempts the state to state transition from this actin the following resistive load the lead the trailing head is its and the lead head its and as the trailing head, one actin (i.e. to the leading head This in the of that are most and the motor to state the state predominantly under load. In the presence of Pi the of and the that in the of Pi to detachment We the of to load, from one step in under to one step in two at This load-dependent equilibrium backward to forward steps to the backward stepping as a of load where is the of the attached in This that a load of The and load of this is to that backstepping in the of ATP J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar) and of single heads T.J. Sweeney H.L. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13873-13878Crossref PubMed Scopus (147) Google Scholar). it is that resistive loads detach the lead head from a conformation. this we have used both load and Pi to the of myosin Our a model in which myosin V can through two pathways in a branched and that the flux through these pathways is by load and Pi. The flux through these pathways be by the of Pi release from the leading head state to the of ADP release state from the trailing The this but that Pi release along A is slow under (15Baker J.E. Krementsova E.B. Kennedy G.G. Armstrong A. Trybus K.M. Warshaw D.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5542-5546Crossref PubMed Scopus (139) Google Scholar), flux through The of Pi release in both and double-headed myosin V (10De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar, 12Rosenfeld S.S. Sweeney H.L. J. Biol. Chem. 2004; 279: 40100-40111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) the flux through of these biochemical the of Pi release from the leading head the trailing head is strongly bound to actin ADP in the active the state state this is the of this alternate be a of At high the of increases load the of the lead head from may myosin V to multiple to and the of actin to alternate myosin V as two a the heads so that myosin V can deliver its cargo the to it by the cytoskeletal We Krementsova and the myosin V Kennedy from the and at the of and and the Trybus and the Warshaw We the of and at the of this so leading to and of which was to was is
Kad et al. (Mon,) conducted a other in Myosin V kinetics. Applied load and phosphate (Pi) vs. Absence of Pi / different loads was evaluated on Myosin V stepping kinetics (forward/backward steps and detachment). Applied load and 40 mM phosphate act as checkpoints that control the flux of myosin V through two parallel kinetic pathways by altering forward and backward stepping probabilities.