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Non Print Items

Abstract
Microscopic reversibility and its corollary, detailed balance, are perhaps the most important fundamental principles necessary for understanding the mechanism by which molecular motors and pumps use chemical energy to perform osmotic, electrical, and/or mechanical work. Despite their centrality however, there is a great deal of confusion in the literature about these principles. In this chapter I will review microscopic reversibility and detailed balance as applied to proteins in general, but specically focussed on application to molecular motors and pumps.

Keywords
AU1 Detailed balance; Microscopic reversibility; Molecular motor; Molecular pump.

Author and Co-author Contact Information

R.D. Astumian Department of Physics and Astronomy University of Maine Orano, ME USA Tel.: (207)581-1024 Fax: (207)581-3410

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4.2 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
RD Astumian, University of Maine, Orano, ME, USA
r 2012 Elsevier Inc. All rights reserved.

Glossary
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4.2.1

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Equilibrium There are several different types of equilibrium relevant for molecular motors and pumps. First, thermal equilibrium implies that the temperature of the system is the same as the temperature of the environment. Statistical equilibrium implies that the state probabilities (or densities) are given by a Boltzmann distribution. Finally, mechanical equilibrium implies that acceleration is unimportant and hence that the velocities are proportional to the forces that cause them a condition that holds if the viscous drag is sufciently large. Microscopic reversibility A fundamental principle for microscopic systems whereby the conditional probability for a trajectory divided by the conditional probability for the exact reverse of that trajectory is the exponential of the difference of the internal energies at the two endpoints plus/minus the work done on/by the environment in following the trajectory. Detailed balance The principle by which the ratio of the forward and backward rate constants for a chemical process is equal the equilibrium constant for that process. Detailed balance is very similar to, and indeed a corollary of, the principle of microscopic reversibility.

Scallop theorem A principle formulated by Purcell4 by which reciprocal motion a cycle of motion where the backward movement follows the identical path that the forward motion took can never cause net displacement. Internal (endogenous) noise Fluctuations of thermodynamic parameters pressure, temperature, electric eld strength, etc. that arise from processes internal to the system of interest. External (autonomous) noise Fluctuations of thermodynamic parameters - pressure, temperature, electric eld strength, etc. that arise from some external power source or noise generator. Dynamic disorder Any uctuations, including internal and external noise, that inuences the behavior of a kinetic system and where the time scale for the uctuation is neither much shorter than, nor much longer than, the characteristic time scale for the kinetic system that is inuenced. External noise can be modeled simply with a equation of motion for the noise parameter that does not depend on the state of the kinetic system. Internal noise, however, is intrinsically coupled to the system and this must be reected in the equation of motion for the noise parameter.

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doi:10.1016/B978-0-08-095718-0.00402-8

4.2.1 4.2.2 4.2.3 4.2.3.1 4.2.3.2 4.2.4 4.2.4.1 4.2.4.2 4.2.4.3 4.2.5 4.2.6 4.2.7 4.2.8 References

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Introduction - Molecular Motors at and Away from Equilibrium Microscopic Reversibility and Ligand Binding to Myoglobin Cycles of Molecular Machines Pumping Catalysis Molecular Machines in a Thermal Environment Pumping a chemical system Catalysis in a chemical system Selectivity and Specicity How Does the Chemical Reaction Drive Conformational Cycling? A Two-Headed Motor that Walks Down a Track Microscopic Reversibility and Dynamic Disorder Conclusion

1 3 5 6 6 6 8 9 9 10 10 11 12 12

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Introduction - Molecular Motors at and Away from Equilibrium

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The goal of this chapter is to set the stage for a theoretical understanding of molecular motors, and specically to illuminate how energy derived from the hydrolysis of ATP at the active site of a protein is able to cause that protein to move along a bio-polymeric track in a preferred direction, even against an externally applied opposing force. To begin on a solid footing let us consider rst a molecular motor, e.g.

kinesin a two-headed protein that uses ATP energy to walk down its microtubule track at chemical equilibrium where the chemical potential of ATP is equal the sum of the chemical potentials of ADP and Pi. Equilibrium for a molecule is not static, but rather a dynamic state in which all possible motions occur, each motion being counterbalanced on average by the microscopic reverse of that motion1. Thus, at chemical equilibrium in the absence of external forces, kinesin continually steps forward and backward with equal probability, and, also with equal probability, hydrolyses and synthesizes ATP. Over a

Comprehensive Biophysics, Volume 0

BIPH 00402
2 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps

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time period that is long compared to the average cycle time, t, of the motor molecule (about a millisecond) the motor molecule does not move preferentially one way or the other on its track, nor is there any net conversion between ATP and ADP and Pi. When ATP is present in excess (i.e., when mATP4mADP mPi) the system is no longer in equilibrium, and the individual motor molecule walks unidirectionally along the polymeric track while catalyzing the net conversion of ATP to ADP and Pi. We are forced to ask ourselves what is different when ATP in the bulk is out of equilibrium with ADP and Pi in the bulk? Are the states accessible to the protein different than when the overall system is in equilibrium? Certainly not an individual motor molecule has no way of sensing the bulk concentration of ATP or of ADP. Similarly, the character of the motion by which a protein undergoes transition from one state to another is no different when the system is away from equilibrium than than when the system is at chemical equilibrium. Indeed the only logically possible difference is that, when the active site is unoccupied, the relative probability to bind ATP vs. ADP away from equilibrium is different than the relative probability when the system is at equilbrium. This seemingly small difference is sufcient to result in almost deterministic stepping when the difference in chemical potential, Dm mATP (mADP mpi) is much greater than the thermal energy, kBT. Under physiological conditions DmE20kBT. We can understand how this is the case by appeal to the principle of microscopic reversibility. A fundamental mathematical expression resulting from microscopic reversibility, and which is valid for all isothermal molecular systems, can be simply derived2 from the thermodynamic action principle of Onsager and Machlup:3 is

p0035

rAPB; tf 9?9A; ti rDPA; tf 9?z9D; ti holds ONLY at statistical equilibrium, that is where the densities, r(A) and r(D) are given by a Boltzmann. However relation, eqn [1], derived2 using the Onsager-Machlup thermodynamic action theory, holds when the system is in equilibrium in the sense required by Onsager and Machlup, that is whenever the velocity is proportional to the forces that cause it3. For single molecules this is nothing other than the condition of low Reynolds number4 where the inertial force (mass times acceleration) is negligible in comparison to the viscous drag force (coefcient of viscous friction times velocity), and hence where the velocity is proportional to the sum of the electrical, mechanical, and chemical forces. These different senses of equilibrium (for a discussion see5) cause great confusion in the literature where it is very easy to dismiss theories of biological processes based on microscopic reversibility (eqn [1]) or the Onsager-Machlup theory by the glib assertion that these theories are valid only very near equilibrium. The equilibrium required for eqn [1] to be valid no acceleration holds for all single molecules in solution on times scales longer than a few picoseconds! Two other key identities can be easily derived from the microscopic reversibility relation eqn [1]. First, we obtain an identity for the exponential average of the work in terms of the internal energy change. Observe the normalization condition for all trajectories, s, between A and D, and between D and A, Z Z PD; tf 9y9A; ti ds PA; tf 9yz9D; ti ds 1:

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S s s

O F

p0040

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S

PD; tf 9?9A; ti e PA; tf 9?z9D; ti

DUAB W kB T

Using this normalization we immediately nd, using eqn [1], that: D w E UAD e kB T : 2 kB T e Second, we obtain a simple relationship between the probability for work W to be done in a trajectory from A to D, and the probability for work -W to be done in a trajectory from D to A. The probabilities for the work to be precisely W for a trajectory from A to D, and -W in a trajectory from D to A, are: Z PA-D W dW WyPD; tf 9y9A; ti ds PD-A W Z dW WyzPA; tf 9yz9D; ti ds

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Equation [1], in words, states that the ratio of the probability, given that the system (e.g., the protein) starts in some conguration A at some initial time ti, of undergoing a transition by the specic pathway (indicated by y) to a conguration D at some nal time tf to the probability, given that the system starts in some conguration D at time ti, to undergo a transition by the microscopic reverse of the specic pathway (indicated by yz) to a conguration A at time tf is the exponential of the sum of the difference in the internal energy of congurations A and D, DUAD, and the work, W(y), exchanged between the system and the environment. The internal energy difference, DUAD, is a state function that depends only on the endpoints A and D. The work, W(y) W(yz), however, depends on the specic path and is the sum of the chemical, electrical, mechanical, and any other, work done on/by the environment in the transition A-y-D. Equation [1] is very general and follows from the microscopic reversibility of the dynamics of any isothermal molecular system. It is often stated that microscopic reversibility holds only at equilibrium. Indeed, it is true that the relation:

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Using eqn [1], the fact that W(yz) W(y), and the fact that d( x) (x)we take the ratio of the two probabilities to obtain:
DUAD W PA-D W e kB T PD-A W

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3
p0060

Equations [1], [2] and [3] could be described as nonequilibrium relations since the densities r(A) and r(D) do not have to be given by a Boltzmann distribution in order for these equations to be valid. Equation [1] does require however

BIPH 00402
Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
that the system is in equilibrium in the Onsager sense; in which acceleration can be ignored and where the velocity is proportional to the force that causes it3. The validity of equations. [1], [2] and [3] under far from statistical equilibrium conditions highlights what has been described as the unreasonable effectiveness of equilibrium theory for describing non-equilibrium systems6. As a specic example of how eqn [1] can be applied to molecular motors, let us take a simple model where A represents a state of the motor protein in which no ligand is bound to the motor, and in which both heads are attached to the track. D represents a state in which one ligand molecule (be it ATP or ADP) is bound and in which the head to which ligand bound is detached from the track. Then the processes in which A undergoes transition to D can be written as: Pf 1 PD9bind ATP to rear head9A Pb1 PD9bind ATP to front head9A Pb2R PD9bind ADP to rear head9A Pf 2R PD9bind ADP to front head9A:

p0065

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Pf 1R PA9release ATP from rear head9B Pb1R PA9release ATP from front head9B Pb2 PA9release ADP from rear head9B Pf 2 PA9release ADP from front head9B:

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The times ti and tf are arbitrary and so we do not write them explicitly. Using these elementary processes we can write joint conditional probabilities for the process, F , in which one ATP is hydrolyzed and the motor takes a step forward as PF Pf1Pf2; for a process, B, in which one ATP is hydrolyzed and the motor takes one step backward as PB Pb1Pb2; for a process, C, in which one ATP is hydrolyzed and the motor does not take a step in either direction as PC Pf1Pb2 Pb1Pf2; and for a process, S, in which no ATP is hydrolyzed but the motor takes a step forward as PS Pf 1 Pb1R Pb1 Pf 2R : The probability for the reverse of each composite process, PF R ; PBR ; PCR ; PS R is obtained by using the probability of the reverse of each of the elementary processes of which it is composed, e.g., PF R Pf 1R Pf 2R : The total work for each composite process is W(F ) Dm Fl, W(B) Dm Fl,W(C) Dm, and W(S) Fl, where F is the external force and l the step length. Using eqn [1] we obtain the general relations:

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The microscopic reverse processes in which D undergoes transition to become A can be written, respectively, as:

where PF and t, the characteristic cycle time for the motor, may depend on the applied force in a way not constrained by microscopic reversibility. Note that for large Dm the predominate mechanism for stepping backward, B, involves hydrolysis, not synthesis, of ATP and is not the microscopic reverse of the predominate mechanism for stepping forward F , a fact that is consistent with the model of Astumian and Bier7. These researchers predicted that as an external force is applied, the system responds by changing the stoichiometry. Interestingly, a large applied force actually stimulates ATP hydrolysis. Thia predicted behavior was experimentally observed by Carter and Cross8 who showed that back-stepping in the presence of a large rear-directed external force is stimulated rather than inhibited by ATP. A second key observation is that, in the absence of an external force, the ratio of forward-to-backward steps, PF =PB with forward chemistry (Dm40) is exactly the same as the ratio of backward-to-forward steps, PF R =PBR with backward chemistry (Dmo0). Thus, it cannot be protein structure alone that dictates the relative probabilities for stepping forward and backward. From this introductory discussion it should be clear that a thorough and deep understanding of the physics behind the principle of microscopic reversibility and how it to pertains to proteins is essential to the development of a theory for biomolecular machines. Thus we will rst discuss microscopic reversibility and simple ligand binding (O2) to a protein myoglobin. From there we will see how despite microscopic reversibility, the cyclic motion necessary for the function of a molecular machine can be driven in a thermal environment by pumping with an external time-dependent perturbation or by catalysis of an energy releasing chemical reaction (e.g., ATP hydrolysis). Then we will discuss how we can modify current theoretical descriptions of the effects of uctuations (sometimes called dynamic disorder) on proteins to be consistent with microscopic reversibility, and nally we summarize our conclusions and contrast the picture of molecular motors developed in the light of microscopic reversibility with standard models involving localized energy release used to drive a power-stroke.

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Microscopic Reversibility and Ligand Binding to Myoglobin

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DmFl DmFl Dm Fl PF PB PC PS e kB T ; e kB T ; ekB T ; ekB T PF R PBR PC R PS R

The motor velocity, v, and rate of ATP hydrolysis, r, can be written in terms of the ratios of PB ; PS , and PC , to PF ,   P   P  ! lP DmFl DmFl Fl B S F v 1 e kB T 1 e kB T 1 ekB T PF PF t r  1e  DmFl
kB T

 P  ! P DmFl Dm PB  C F 1 e kB T 1 e kB T PF PF t

Myoglobin is one of the best studied proteins9,10, and one of the rst for which the X-ray crystal structure was determined11. When oxygen or carbon monoxide bind to the heme-group of myoglobin, the heme undergoes a transition from a conguration in which the iron atom is out of the plane of the heme to a conguration in which the iron is in-plane. The local congurational change is followed by a large scale conformation change of the protein. This mechanism for the binding reaction can be summarized as: ! Mbop O2 -Mbip O2 -Mbip O2 Mbop
O2

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BIPH 00402
4 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
state has occurred, follows very naturally from macroscopic analogy. If we imagine that we insert our nger into a calm body of water, we see ripples propagating from our nger outward. After the water has returned to quiescence, when we remove our nger the ripples once again propagate outward from where our nger had been. This picture was explicitly suggested by Ansari et al.12 for ligand association/dissociation to myoglobin. These authors stated that binding or dissociation of a ligand at the heme iron causes a protein-quake, with the heme as the focus of the quake. However, such a picture is not consistent with microscopic reversibility, and is possible only in the case of photochemically induced dissociation. If thermally activated binding causes a quake propagating outward from the focus, then, counter-intuitive though it may be, thermally activated dissociation must arise by an inward propagating un-quake that triggers the release of the ligand. Conformational transitions - shape changes - of a protein (or any other deformable body4,13) cause the center of mass of the protein to move relative to the uid in which the protein is immersed. A combination of any set of transitions constrained by microscopic reversibility such that the backward reaction (e.g., mechanism eqn [6]) is the microscopic reverse of the forward reaction (e.g., eqn [4])

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These two different mechanisms for the dissociation of a ligand from the heme group, depending on whether the reaction occurs by photolysis or by thermal activation, conform with the application of the principle of microscopic reversibility as dened in the International Union of Pure and Applied Chemists (IUPAC) Compendium of Chemical Terminology (http://goldbook.iupac.org/), known informally as the Gold Book:
Microscopic Reversibility - In a reversible reaction, the mechanism in one direction is exactly the reverse of the mechanism in the other direction. This does not apply to reactions that begin with a photochemical excitation.

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Mbip O2 -Mbip O2 -Mbop O2 - Mbop

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This mechanism is clearly not the microscopic reverse of the reaction by which binding of oxygen occurs. For nonphotochemically assisted dissociation, however, according to microscopic reversibility we must have for the most probable pathway the microscopic reverse of the binding reaction, that is for thermally activated dissociation we have the mechanism:

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where the conguration with the iron out of plane is denoted by subscript op, and that where the iron is in plane is denoted by the subscript ip. The two different global conformational states are denoted by italic font ( Mb) for the oxygenated form, and by Roman font (Mb) for the deoxygenated form. When we think about the reverse of the binding process, we are tempted to imagine a scenario in which oxygen dissociates followed by return of the heme group to its original out-of-plane conguration, and subsequently a global rearrangement of the molecule to restore the initial equilibrium conformation for the protein where the binding site is unoccupied. Indeed, this picture seems to be well supported by experiments on ligand dissociation from myoglobin, where a ligand is caused to dissociate at low temperature by a LASER pulse, and the subsequent conformational relaxation is studied. These investigations reveal that after light-induced dissociation the myoglobin molecule undergoes a local rearrangement followed by a global conformational change in what has been termed a protein quake12. The mechanism for photolytic dissociation can be written as:

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This denition follows from eqn [1]. There are two possible values of the work W for dissociation. For the thermally activated process Wth mO2 , and for the photochemical process Wphoto hn mO2 : Thus, thermally activated binding is (almost innitely) more probable than binding with the concommitant emission of a photon, while, in the presence of light, dissociation and absorption of a photon is more likely than thermally activated dissociation without absorption of a photon. The idea that, following either binding or dissociation of a ligand at the heme, the conformational rearrangement of the protein starts locally and propagates through the protein until the global change to the new equilibrium conformational

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gives rise to a reciprocal process that according to Purcells scallop theorem4 cannot, in the absence of inertia, cause net directed motion in a cycle of the forward and backward transitions. The scallop theorem is so named because the opening and closing of a scallop shell occurs on a single coordinate or degree of freedom. At low Reynolds number, where viscous drag dominates inertial force, such back and forth motion cannot lead to a net displacement because there is no glide, and whatever is done in the forward process is undone in the backward process. Thus, the thermally activated binding/release of oxygen or carbon monoxide to myoglobin does not provide, even in principle, a mechanism for propulsion of the protein through solution. On the other hand, although there are doubtless many practical reasons that are not biologically relevant for myoglobin, there is no fundamental reason that a cycle of thermally activated binding (eqn [4]) and photochemically induced dissocation of a ligand, eqn [5], where the forward conformational change is not the microscopic reverse of the backward conformational change) could not provide an effective mechanism for propulsion under the right circumstances. The combination of eqn [1] and [2] can be written as:
Mb ip h , O2 Mb ipO2 deoxyMbop

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where it is apparent that the conformational relaxation following photo-assisted dissociation is not the microscopic

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Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
reverse of the conformational relaxation following thermally activated binding. As a matter of principle, any conformational cycle of a protein or macromolecule (or anything else) in viscous solution A-B-?-D-?-F-A can, and in general will, lead to directed motion13, whether it be of an ion across a membrane, stepping along a polymeric track, or self-propulsion through the aqueous solvent. Further, completion of the cycle backward A-F-?-D-?-B-A will cause exactly the reverse of the motion caused by the forward cycle. In the Myoglobin cycle eqn [5], the two stable states are A Mbop and D MbipO2. Let us now consider how such non-reciprocal cyclical processes can occur without photochemical activation.
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4.2.3

Cycles of Molecular Machines

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In a recent paper, Togashi and Mikhailov14 proposed that a macromolecule, modeled as an elastic network, can be constructed to operate as a cyclic machine powered by ligand binding. The binding was modeled by forming elastic links between the ligand and nearby nodes of the elastic network, and allowing the network to relax to its new conformational energy minimum. The ligand was then removed (the elastic interactions were deleted) and the system again underwent conformational relaxation. The overall process resulting from adding ligand, relaxation, removing ligand, relaxation, adding ligand, etc. was described by a simple cycle (Figure 1):
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Figure 1 Energy diagram for the binding/dissociation reactions of myoglobin. In photo-dissociation the photon energy promotes the molecule to a very high energy state Mbip from which it undergoes non-equilibrium functionally important motions to reach the deoxygenated ground state deoxyMbip In contrast, thermally activated dissociation must follow the reverse path (denoted by the dashed arrows) of the thermally activated binding process according to the principle of microscopic reversibility. The transitions between the states are all equilibrium-uctuations (including those that may be functionally important) in that they occur by exactly the same physical mechanism both at and away from equilbrium.

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Figure 2 Energy levels for binding and dissociation of ligand, L, to a polymer with several conformational states, where the lowest energy conformation with ligand at the binding site (state DL) is different than the lowest energy conformation without ligand at the binding site (state A). Obviously the transition DL-E requires a great deal of energy. From where does this energy come? In the computation it comes from the enforced removal of the ligand, but how could this energy be supplied in a physical system absent a photochemical process. Two possibilities are pumping (Figure 3) and catalysis (Figure 4). f0015

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This proposed mechanism for autonomous generation of non-reciprocal cyclic motion is not consistent with microscopic reversibility if the ligand that binds to state A is the same molecule as the ligand that dissociates from state DL and under the same conditions. We can understand why this is the case by an energetic analysis (Figure 2). Clearly, the energy of state A is less than that of state E, and the energy of state DL is less than that of state BL. No matter how we arrange the energies of the bound states relative to the energies of the non-bound states, the overall process of binding a ligand to A and releasing a ligand from D needs energy (at least (eE eA) (eB eD)), which cannot be provided by the binding and release of the same ligand under the same conditions. In Togashi and Mikhailovs work, the ligand dissociating from the macromolecule was implicitly different than the ligand that had associated. In the computational experiment the energy released by the chemical conversion was added by the way the binding and release of ligand was modeled - the elastic network was allowed to relax to the minimum energy

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conformation (A) in the absence of ligand, and then the ligand was instantaneously inserted by adding elastic interactions, placing the system in state BL . The system, under these new conditions with a ligand at the binding site, was no longer at a minimum energy conformation, and so relaxed to the minimum energy conformation with ligand bound (DL). Then, the ligand was instantaneously removed by eliminating all interactions with the elastic network, thus placing the system in state E, and the subsequent conformational relaxation to the original energy minimum conformation (A) with no ligand at the binding site was followed. Thermal noise was not included in the computational study, and the transitions BL-CL-DL and E-F-A were deterministic overdamped elastic relaxation processes. The details of the ligand

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6 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps 4.2.3.2 Catalysis

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binding A-BL and dissociation DL-E were not explicitly discussed, nor was the role of the chemical potential of the ligand. The energy differences DeEF and DeFA for the unbound states were calculated directly from the elastic network model, as were the energy differences DeBC and DeCD for the bound states. In contrast, the energy differences between states A and 0 B, DeAB DeAB mL, and between states D and F, 0 DeDF DeDF mL, must depend on the internal energies of states A and B, and D and F, respectively, and on the chemical 0 potential of ligand in the bulk, that is , DeAB DeAB mL and DeDe De0 mL where mL m0 RT ln (aL) is the chemical De L potential of the ligand given as the sum of a standard chemical potential and the activity, aL of the ligand. In dilute solution, the activity is the concentration divided by a reference concentration of 1 mol/liter and is hence dimensionless. The derivation of the energy difference between the bound states and the non-bound states immediately suggests two approaches for how to use ligand binding and dissociation to drive non-reciprocal cycling of the macromolecule conformational states pumping and catalysis.

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A second mechanism by which conformational cycling can be driven is catalysis. If, while bound to the macromolecule, the ligand L, at concentration [L]4KL can be catalytically cond verted to some different product, P, at concentration [P]oKP d we can have a situation where De0 mL40 and also where AB De0 mP40. DF Then, both binding of L to A, and dissociation of P from DL are spontaneous. The macromolecule will carry out conformational cycling so long as the chemical concentrations of L and P are maintained at these levels. The energy provided by each conversion of L to P is:   L ein mL mP Dm0 RTln LP P

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which can be used to power conformational cycling.

4.2.4

Molecular Machines in a Thermal Environment

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4.2.3.1

Pumping

?-A - BL -CL -DL - E-F-A-?:

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LooKd

p0185

This mechanism requires external oscillation or uctuation of the ligand concentration, although in principle, if the ligand were some intermediate in an oscillating chemical reaction16 the process could be driven without direct experimental manipulation of the concentrations. The pumped energy ux into the system that allows work to be done on the environment comes from the fact that, on average, the ligand is bound while the chemical potential is high, and dissociates when the chemical potential is low. Through a cycle of oscillation of the ligand concentration, the energy available to drive ux through the conformational cycle is, at most, equal the amplitude of the oscillation of the chemical potential:   Lhigh ein mLhigh mLlow RT ln Llow and is hence the maximum work that can be done on the environment.

EL

SE

VI

ER

FI

p0175

We can pump non-reciprocal conformational cycling15 by externally driven oscillations or uctuations of the concentration of the ligand between a high and low level. The high level [L]highcKd, favors binding of ligand to A followed by elastic relaxation BL-CL-DL, and the low level [L]Low{Kd favors dissociation of ligand from DL followed by elastic relaxation E-F-A. The dissociation constant Kd is that concentration at which half the macromolecule is bound to a ligand half is not bound. The oscillation (or uctuation) of the concentration of L can be repeated, resulting in continual cycling:

Ultimately, our goal is to understand how molecular motors convert chemical energy into non-reciprocal conformational cycling, and hence into directed motion and mechanical work, in solution at room temperature where thermal noise is very strong. As a result of thermal noise, there is a continual, reversible exchange of energy between each macromolecule molecule and its environment. This fact has extremely important ramications for how we should describe and think about molecular motors. When we look at the mechanism in Figure 2 it is tempting to term the elastic relaxation processes BL-CL-DL and E-F-A as power strokes, and indeed they are - power is dissipated as the system undergoes elastic relaxation. In a thermal environment, though, we can compare the power dissipated during the power stroke with the power that is continually and reversibly exchanged between the macromolecule and the environment to gauge the relative importance of mechanical vs. thermal effects. If at some point on its energy prole the macromolecule experiences a very large force of 100 pN that at that instant moves the center of mass of the macromolecule with the very large velocity of 1 m/s, the power instantaneously dissipated by the power stroke is Pdiss 100 pN 1 m/s 1010W. In contrast, the power reversibly exchanged with the environment at room temperature is KBT 4 1021J every thermal relaxation time tthE1012 S or 4 109W 40 times greater than the maximum power dissipated during even a very powerful molecular power stroke!5 Further, as recognized by Andrew Huxley17, the reverse, or un-power strokes DL-CL-BL and A-F-E in which energy ow from the environment to the protein will also occur at appreciable rates. In contrast to the patently nonequilibrium functionally important motions following photo-dissociation of oxygen or carbon monoxide from myoglobin12, the conformational changes by which chemically driven molecular motors move are purely equilibrium processes. The only difference between equilibrium and non-equilibrium for a thermally activated mechanism has to

PR

p0200

ST

p0205

BIPH 00402
Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps 7

L E F A BL CL DL

+L

m at io na ls el ec tio n

BL CL DL

(L) >> Kd

(L) << Kd

E F A

C on fo r

kEF kFE

kFA kAF kBA

A (L)kAB

f0020

Figure 3 Pumping mechanism for driving conformational cycling. By externally changing the ligand concentration between concentrations where the macromolecule is most likely bound and concentrations where the macromolecule is most likely not bound we induce a situation where the ligand binds at high chemical potential and dissociates at low chemical potential, dissipating kBT ln ([L]high/ [L]low) energy with each binding-dissociation event. Some of this energy can be harnessed to drive directional cycling, and even to do work on the environment.

kDE

(L)k ED

kCD

kBC

+L BL CL DL

P E F A

+L BL CL DL
f0025

change, induced t and conformational selection, respectively18 (Figure 5). A second entry for microscopic reversibility in the IUPAC Gold Book denes:

PR

O
eq kij ci

Figure 5 Illustration of a kinetic cycle mechanism where there are two paths between the states A and DL, one in which the ligand binds and then conformational relaxation occurs, known as an induced t mechanism, and the other in which the conformational change occurs, followed by binding, known as a conformational selection mechanism. In the usual way of considering conformational selection vs. induced t focuses on ligand binding and not on dissociation. However, by microscopic reversibility if the induced t mechanism is preferred when a ligand binds, under identical conditions the reverse of that process must also be preferred when ligand dissociates.

In du ce d

DL

kDC

CL

kCB

BL

fit

f0030

O F
eq kji cj

p0220

AU5

p0210

EL

p0215

do with the binding of the ligand. The physical motions of the molecule that follow binding, or dissociation of a ligand, are exactly the same at and away from equilibrium (Figures 3 and 4). Far from being predominately mechanical devices slightly perturbed by thermal noise, molecular motors are overwhelmingly dominated by thermal effects, that is molecular motors are rst and foremost molecules, and they operate based on the laws of chemistry rather than those of macroscopic mechanics. In order to capture the effects due to interaction with the thermal environment, we must consider a chemical kinetic mechanism with forward and backward reactions (Figure 5). In this system, for each transition i-j there is a rate constant, kij, expressing the probability per unit time for that transition to occur given that the system is in state i. The effective rst 0 order rate constants for the binding reactions kAB LkAB and 0 kED LkED are the product of the ligand concentration and a bimolecular rate constant. It is interesting to note that the two pathways between A and DL, A-BL-CL-DL and A-F-E-DL correspond to the two well known limiting mechanisms for ligand binding coupled to conformational

ER

Figure 4 Catalytic mechanism for driving non-reciprocal conformational cycling. If the macromolecule facilitates conversion of L to P, where the concentrations are maintained such that [L]4KL d and [P]oKP both binding of L and release of P are spontaneous d processes that, coupled with the elastic relaxation of the macromolecule network allow net conformational cycling. The free energy provided by each conversion (and hence available to drive   L cycling) is. Dm0 RT ln P . LP

FI

ST

The principle of microscopic reversibility at equilibrium states that, in a system at equilibrium, any molecular process and the reverse of that process occur, on the average, at the same rate. This denition corresponds to the statement of the principle that was given by R.C. Tolman in 1924. However, many workers have interchanged the meanings of microscopic reversibility and detailed balance, and it seems best now to regard the two, which are closely related, as synonymous.

Thus, at equilibrium we must have 0; 9

p0225

SE

VI

where ceq denotes the fractional concentration in state i at i equilibrium (concentration of macromolecule in state i divided by the total macromolecule concentration). The ratios of the rate constants are the equilibrium constants: kij Kij e kji DGij mL kB T

ij AB; ED ij BC; CD; EF; FA

DGij kij Kij e kB T kji

10

From eqns [9] or [10] it is easy to derive a relationship between the product of the clockwise rate constants and the product of the counter clockwise rate constants. In the absence of external vectorial elds or forces: kAB kBC kCD kDE kEF kFA 1 kAF kFE kED kDC kCB kBA 11

p0230

BIPH 00402
8 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
back to A. The ligand will then dissociate from the macromolecule in state DL making a transition to state E, from which it relaxes to state A through state F, thus completing a clockwise conformation cycle through the states. Clearly, since [L] cancels in the numerator and denominator of eqn [11] or eqn [12], if the system is not set up to do work on the environment, eqn [11] holds at every instant in time. Further, if the conformation cycle is set to do work on the environment (so that eqn [12] rather than eqn [11] holds), pumping can drive clockwise ux to do work on the environment, with a theoretical limit of W mLhigh mLlow . For realistic parameters the efciency - the ratio of output work to input energy - can be very near to one15. In a thermal environment there is of course a nite probability that when the concentration is switched from high to low, the molecule will be in state BL rather than state DL, and when the concentration is switched from low to high, the macromolecule will be in state E rather than in state A. If this happens the oscillation of the ligand concentration will cause counter-clockwise rather than clockwise conformational cycling. We can easily estimate the relative likelihood for this to occur if switching ligand concentration between very low ([L]{Kd) and very high ([L]cKd) levels is accomplished very rapidly, and the system is allowed to reach equilibrium after each switch, in which case the ratio of the probability to complete a clockwise (P ) vs. the probability to complete a counterclockwise cycle (P_) through the conformational states is15

p0235

If the cycle is set up in the presence of time independent elds, or forces such that completion of the cycle in the clockwise direction does work on the environment, W, some of the rate and equilibrium constants must be modied. Although the details of which rate and equilibrium constants change and by how much are model specic, the relation eqn [11] between the products of forward and reverse rate constants can be generalized to read:
W kAB kBC kCD kDE kEF kFA e kB T kAF kFE kED kDC kCB kBA

12

p0240

s0040 p0245

4.2.4.1

Pumping a chemical system

p0250

There is much confusion in the literature concerning what eqn [11] does and does not mean with regard to driving cyclic ux. It is often asserted that the relation eqn [11] is a necessary and sufcient condition for a system to be in equilibrium,19 or that the system is in equilibrium if and only if eqn [11] holds,20 or that the cyclic ux is zero if eqn [11] holds,21 or that cyclic ux is possible if and only if eqn [11] does not hold.22 These, and other similar statements, while valid for time-independent rate coefcients, fail to take into account the possibility of pumping by externally driven oscillations23 or uctuations24 of some thermodynamic parameter such as pressure, electric eld strength (particularly relevant for membrane transporters), or ligand concentration. In the context of pumping an electrically neutral substrate across a membrane by an oscillating membrane potential, Robertson and Astumian25 explicitly pointed out that when an AC eld is switched on, a necessary condition for detailed balance (eqn [11]) continues to hold at every instant in time. Nevertheless, the eld causes net clockwise ux. The oscillating conditions coupled to the conformational transitions of the protein allow energy to ow into the system from the source of the oscillations to drive directed cycling.26,27 Let us now consider how pumping the ligand concentration can drive a macromolecule through a non-reciprocal conformational cycle in a thermal environment. Let the concentration of ligand start out very low relative to the dissociation constant, [L]{Kd. The macromolecule will almost certainly be in state A. Then, we rapidly increase the ligand concentration, lowering the energy levels of the bound states BL,CL, and DL relative to the free states E, F, and A. The macromolecule in state A will then almost certainly bind a ligand to make a transition to state BL, after which it will undergo conformational relaxation through state CL until equilibrating in state DL. Then we rapidly lower the ligand concentration, thereby increasing the energy levels of the bound states BL,CL, and DL relative to the free states E, F, and

ST

PR

O
IFA
Pump

Equation [12], derived from knowledge that the system obeys the principle of microscopic reversibility as formulated in eqn [1] holds whether the system is or is not at statistical equilibrium with regard to the concentrations. Equation [1] is the fundamental equation expressing microscopic reversibility and focues on the ratio between the conditional probabilities of a trajectory and its microscopic reverse. Equation [12] is based on the detailed balance for transitions between discrete states of a system.

p0255

DGEA DGBD W P e kB T e kB T P

O F
dcA : dt

13
p0260

EL

The essential mechanism is that of an energy ratchet28 as soon as L binds when [L]cKd the macromolecule relaxes from state BL to state DL becauseDGBD40, and as soon as L dissociates when [L]{Kd the macromolecule relaxes from state E to state A because DGEA40. With two externally controlled parameters (e.g., pH and redox potential) it is possible to achieve pumping and directional conformational cycling with oscillations carried out so slowly that the fractional concentrations of the states have their equilibrium values, and hence eqn [9] hold at every instant in time.29 On the molecular time scale (where the relaxation time may be on the order of microseconds) very slow can be up to several thousand cycles per second, an appreciable rate from the macroscopic perspective, and consistent with rates observed for biomolecular motors. In recognition of the ineluctable presence of uctuations, the classic pitcher of a steady-state (ss) current, e.g. between states F and A, ISS kFA CSS kAF cSS : F FA A where the overline indicates a time average, must be supplemented with a pumped current30 rAF t

FI

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VI

ER

p0265

p0270

where rAF(t)is the ratio of the ow into A from F to the total ow into A, to get the net current:

BIPH 00402
Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
Inet Iss IFA FA FA
p0275
pump

Equation [11] provides a necessary and sufcient condition for the steady state current to be zero at every instant. However, the pumped current is zero if and only if the inevitable uctuations of rAF (t)are uncorrelated with the uctuations of the rate into/out of state A, dcA/dt. In the absence of external time-dependent ow of energy into the system, this lack of correlation between the two uctuating quantities is assured to hold by microscopic reversibility.

16

s0045 p0280

4.2.4.2

Catalysis in a chemical system

E
P +P L +L

A
P +P L +L

Each cycle, of course, has its microscopic reverse in which the path is traced counterclockwise. The ratio of the probabilities to complete a clockwise vs. counterclockwise cycle for any of the above diagrams is the product of rate constants in the clockwise direction divided by the product of rate constants in the counterclockwise direction, which, using eqns. [14] and [15] works out as:
DmW PF e kB T ; PF R

O
0

The second approach for driving directional cycling is catalysis. If a macromolecule can be designed to catalyze a reaction L"P, then, when the chemical potentials of L and P are not equal, mLamp, the macromolecule will most likely bind whichever has the higher chemical potential, and release whichever has the lower chemical potential. Thus, catalysis autonomously achieves the bind high release low by which pumping drives directional cycling. The mechanism can be written:

O F
p0295

ST

PR

DmW PB e kB T ; P BR

Dm PC ekB T ; PCR

w PS ekB T : 17 PS R

p0285

Dm kL kP kL kP AB BA ED DE ekB T kL kP kL kP DE ED BA AB

ER

Note that, since L and P must be related chemically (i.e., they are interconvertible), either L or P can bind to either state B or D, but with possibly different rates. The effective rst 0 L order rate constants for the binding reactions are kL LkAB AB 0 0 0 L P P and kL LkED , and kP PkAB and kP PkED , and the ED AB ED off rate constants for L and P are kL and kL , and kP and BA DE BA kP . Since the macromolecule acts as a catalyst we have the DE relationship:

14

SE

where Dm mL mP is the difference in the chemical potentials of L and P. We also have eqn [12] which here reads:
w kiAB kBC kCD kiDE kEF kFA ; ekB T kAF kFE kiED kDC kCB kiBA

which is identical to eqn [4] with Fl W. It is through the coupled processes F that chemistry-driven B conformational cycling and the possibility of doing work on the environment arise. For DmcW any backstepping counterclockwise cycling through the conformational state is most likely due to the backward cycle B, in which L is converted to P, rather than to the F R in which P is converted to L. The substrate (L) stimulation of backward cycling was predicted by Astumian and Bier7 and demonstrated experimentally for kinesin (where L ATP) by Carter and Cross8. For W 0, if Dm40 then PF 4PF R and PB 4PBR . Thus, in order to have a molecular machine that drives forward (clockwise) cycling through the conformational states we require only that PF 4PB , that is:
L kAB kP ED 41: 0L kED kP BA

FI

VI

p0300

i L; P

15

EL

p0290

Using Hills approach for the analysis of catalytic kinetic mechanisms in terms of cycles31, scheme (I) can be decomposed into six elementary cycles32 taken in the clockwise sense. Two cycles, C1 and C2, describe conversion of L to P without conformational cycling; two cycles, S1 and S2, describe clockwise conformational cycling uncoupled to conversion of L to P; and one cycle, F , describes clockwise conformational cycling coupled to conversion of L to P. Another cycle, BR , describe clockwise conformational cycling through the states coupled to conversion of P to L:

In order to assure that the motor is effective in moving against a load force, the uncoupled stepping probability PS must also be small, and in order to assure that the motor does not waste chemical fuel, the uncoupled chemical conversion probabilities PC should also be small.

4.2.4.3

Selectivity and Specicity

s0050 p0305

An obvious approach to achieving these goals is to design a macromolecule where the kinetic barrier for binding/dissociation of P from states A/B is much larger than that for

BIPH 00402
10 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
ln([ATP]/[ADP][Pi]B20 kBT) under physiological conditions36. As seen in the discussion above, the idea that energy transduction is a local event is just plain wrong. For thermally activated mechanisms (in contrast to light induced processes like photosynthesis) free energy transduction is a property of the kinetic cycle as a whole.37 There is no single key step, and the free energy available to drive the process is the total free energy change resulting from conversion of reactant in the bulk into product in the bulk. Lauger38 saliently synopsized the mechanism of membrane ion pumps (such as the Na , K ATPase and Ca2 ATPase), observing that
Ion pumps do not function by a power stroke mechanism; instead, pump operation involves transitions between molecular states, each of which is very close to thermal equilibrium with respect to its internal degrees of freedom, even at very large overall driving force.

binding/dissociation of L (i.e., where states A/B are specic for L), and where the kinetic barrier for binding/dissociation of L from states E/D is much larger than that for binding/dissociation of P (i.e., where states E/D are specic for P). The ratios between the on and off rate constants for L and P at equilibrium must be the same k0L Leq =kL k0P Peq ==kP AB BA AB BA and k0L Leq =kL k0P Peq ==kP , where [L]eq/[P]eq Keq LP ED DE ED DE denes the equilibrium constant between L and P. The individual rate constants however can be very different from one another, a reection of different barrier heights, and we can dene specicity parameters in terms of only the off rate constants: sB
p0310

p0330

kL BA ; kP BA

sD

kL DE kP DE

18

s0055

4.2.5

p0320

p0325

The conversion of ligand to product (conversion of ATP to ADP Pi in the case of kinesin and many biological motors) can drive either forward or backward cycling through the states, but with different probabilities. We are hence led to ask the question what exactly does the chemical conversion do?. It is very tempting to think of the mechanism of free-energy transduction in terms of the local action of some energy bringing ligand35 such as ATP which is hydrolyzed at the active site where the released energy is used to actuate a functionally important motion or power stroke that occurs only away from equilibrium and by which directed motion is driven. This idea is represented in animations of kinesin as a ash of light when ATP is hydrolyzed (http:// www.youtube.com/watch?v 686qX5yzksU). Fisher and Kolomeisky explicitly proposed that one should be concerned with the microscopically local release of free energy by ATP adsorbed on the motor-protein track complex and that the free energy available to drive the process is only the basic chemical potential difference, Dm0B12kBT at room temperature, rather than the total concentration-dependent chemical potential difference Dm0 RT

ER

How Does the Chemical Reaction Drive Conformational Cycling?

FI

ST
4.2.6

p0315

Signicantly, this ratio is independent of the free energies of the states, and is hence a chemically driven information ratchet.28,33 The active site acts a Maxwells demon,34 selecting for L in states A/B, and for P in states E/D. Note however that irrespective of how strongly asymmetric the selectivity of the active site is (i.e., if SBcSD or vice versa), the mechanism fails to drive directed transport or to do work in the environment if Dm 0, in consistency with the second law of thermodynamics.

PR

We use eqns [14] and [15] to write the ratio of the overall probabilities for a clockwise ( ) vs. counterclockwise ( ) conformational cycle  Dm  sD 1 sB ekB T 1 W P PF PBR PS   ekB T 19 P PF R PB PSR s 1 s ekDmT 1 B D B

Exactly the same thing could be said of kinesin and myosin. Consider, as an example, a two-headed ATP driven molecular motor that walks along a biopolymer. At chemical equilibrium, where mATP mADP mPi the motor steps forward and backward with equal probability, and hydrolyses and synthesizes ATP with equal probability. Although on average the motor does not move preferentially in one direction, nor does the chemical reaction proceed, on average, to either hydrolyze or synthesize ATP, the equilibrium is maintained dynamically, with each motion being counterbalanced by an equal and opposite motion. When extra ATP is added (or ADP is removed) the character of the motions does not change there is no way for the motor to be directly inuenced by the concentrations in the bulk solution. The only thing that changes is that the probability of binding ATP rather than ADP, when the active site is unoccupied, is increased. We are left with the perhaps unsatisfying but nonetheless accurate answer to the question posed in the section heading the chemical reaction drives conformational cycling by mass action.

VI

A Two-Headed Motor that Walks Down a Track

O F

s0060

In the discussion up to now we have focused solely on the question of how to achieve directional conformational cycling through a sequence of states, with no mention of how this directional cycling leads to net motion, be it transport of some substrate across a membrane, self-propulsion of a molecule through solution, or walking along a polymeric track as in the case of kinesin or myosin. That is because the mechanisms of directional cycling are quite general and based on solid thermodynamic principles32 whereas the analysis of the motion caused by directional cycling is system dependent. For ion pumps, the key features involve gating and afnity switches;39 for self-propulsion at Low Reynolds number a hydrodynamic analysis is essential;40 and for molecules that walk along a track, selectivity switching induced by mechanical linkage through the molecule seem to be a key ingredient for ensuring that ATP hydrolysis produces directional cycling.41,42

EL

SE

p0335

BIPH 00402
Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps
Consider the following schematic mechanism for a two headed molecular motor (represented as ) that walks along a polar polymeric track B__B :
P L P L L P L P

11

p0340

20

p0345

and one step to the - end of the track for each counterclock wise cycle:

s0065

4.2.7

SE

Microscopic Reversibility and Dynamic Disorder

VI

irrespective of whether L or P is bound, and irrespective of whether L or P is released. The kinetic scheme eqn [20] can be broken up into the same six elementary cycles seen in eqn [16] where the ratio of clockwise to counterclockwise cycles is governed solely by the relative specicities and is independent of the free energies of the states. For the specic mechanism of two-headed motion on a track the question is how can the specicity of the two a priori equivalent heads be regulated, such that the rear head predominately binds/dissociates ATP and the front head predominately binds/dissociates ADP and Pi?. A likely mechanism (at least for myosin V) seems to be strain induced gating.41,42

ER

FI

ST

PR

The state represents a motor molecule where neither head is bound to ligand and both heads are bound to the track. Either the rear head or the front head can bind to L or , or P to form the complex respectively. Association with ligand weakens the binding of that head to the track, facilitating the formation of the comwith only one head bound. Eventually the head plex will rebind to the track and L or P will dissociate, reforming . The motor is translated one the original complex step to the end of the track for each cycle in the clockwise direction:

rate constants are obviously not consistent with microscopic reversibility. This thermodynamic inconsistency is a reection of a more widespread incorrect assumption; that it is somehow possible to have an internal environmental parameter that inuences a rate process (e.g., a protein conformational change), but which is not in turn inuenced by the value of the variable described by the rate process. The general approach has been reviewed by Zwanzig45 and used to model escape through a uctuating bottleneck.46 More recently, dynamic disorder has been used to model both protein intermittency47 and enzyme catalysis48 in a uctuating environment. The starting equations are: dc kr c J dt

where c is any physical quantity (e.g., a concentration) that satises a rate equation and r is described as a control variable. The rate constant k(r) is any function of r, e.g., k(r) k0r2 (describing a uctuating bottleneck or gate)46 or kr k0 er=kB T (describing a uctuating energy barrier),48 where k0 and as Gaussian white noise, and there is a uctuation dissipation relation between the amplitude of the noise and the damping coefcient l. This seemingly simple and straightforward approach to modeling the effects of environmental uctuation on a rate process is fundamentally and irretrievably inconsistent with microscopic reversibility. We are supposed to have a control parameter that inuences the dynamics of a physical quantity c, but which is not in turn inuenced by the value of c, a situation that can be descriptive of autonomous (external) noise,24,32 but certainly can not correctly describe the effects of endogenous (internal) noise24 which must be inuenced by feedback. In Figure 6 we show a schematic plot of (r(t), c(t)) parametrized by time, where we take kr k0 er=kB T . The chirality of the trajectory is obvious c must increase when r is less than its average value, and c must decrease when r is greater than its average value. This circulation is a reection of the fact that the vector eld dc ; dr dt dt is NOT curl free! In order to have a consistent model for the effect of uctuations on a rate (or any other physical process), it is necessary to include the reciprocal feedback from c on r.24 This can be accomplished by supplementing the expression for dr=dt, dc krc J dt dr l r f c; r Ft xt dt 22

O F

dr l r xt dt

21

p0360

EL

p0350

p0355

The principle of microscopic reversibility places signicant constraints on the mechanisms by which molecules use free energy to drive directed motion, and to do work on the environment. Of perhaps even greater general importance are the constraints placed on models for the effects of uctuations on all proteins, including enzymes. Recent experimental work has shown that environmental uctuations play a major role in determining the catalytic behavior of enzymes, giving rise to dynamic disorder in the rate constants for the reactions. Several theoretical models based on discrete kinetics fail to respect the constraints imposed by detailed balance. See, e.g., eqn [15] in 44 and eqn [42] and [43] in, 43 where the assigned

where F(t) models the effect of any (possibly time dependent) external driving and we require the coupling function to obey the relationship:49,50

q krc q f c; r 0: qr qc

23

BIPH 00402
12 Microscopic Reversibility and Free-Energy Transduction by Molecular Motors and Pumps

c t

r
f0035 Figure 6 Schematic illustration of a plot of c vs. r parametrized by time for eqn [17] where, e.g., k r k 0 e r =kB T , i.e., k(r) is a decreasing function of r and thus c increases when r is less than its average value and decreases when r is greater than its average value. Obviously there is a sense of time (clockwise cycling) which is not consistent with equilibrium uctuations.

p0365

O F

With this formulation the vector eld dc ; dr is curl free dt dt and hence the uctuation in the joint space (c,r) is time reversible, as it must be, when F(t) 0.

s0070 p0370

4.2.8

Conclusion

ER

It is very tempting to imagine that conformational changes following either ligand binding to or dissociation from a protein start locally and propagate outward from the binding site. We are inuenced by macroscopic intuition to hypothesize that there is some single critical step in which energy, e.g., from ATP hydrolysis, is deposited in a protein, and that a subsequent functionally important motion or power stroke that only occurs away from equilibrium is responsible for the net movement driven by a molecular machine. It further seems reasonable to model the effects of environmental uctuations in terms of some internal environmental parameter that inuences the dynamics of a protein conformational change, but which is not in turn inuenced by the protein conformation. All of these macroscopically inspired ideas are wrong, and are not consistent with the principle of microscopic reversibility.51,1 In formulating models for the operation of molecular machines the mathematical relations, eqns. [1], [12] and eqn [23], derived from microscopic reversibility, protects us from ourselves and from our inability to extrapolate from everyday experience in the macroscopic world to the behavior of molecules in the microscopic world.

bib1 bib2 bib3 bib4 bib5 bib6 bib7 bib8

[1] Astumian, R. D. Kinetics and Thermodynamics of Molecular Motors. Biophys. Jour. 2010, 98, 24012409. [2] Bier, M.; Derenyi, I.; Kostur, M.; Astumian, R. D. Intrawell relaxation of overdamped Brownian particles. Phys. Rev. E. 1999, 59, 64226432. [3] Onsager, L.; Machlup, S. Fluctuatations and irreversible processes. Phys. Rev. 1953, 91, 15051512. [4] Purcell, E. Life at low Reynolds number, Am. J. Phys. 1977, 45, 311. [5] Astumian, R. D.. Design principles for Brownian molecular machines: how to swim in molasses and walk in a hurricane. Phys. Chem. Chem. Phys. 2007, 9, 50675083. [6] Astumian, R. D. Unreasonable effectiveness of equilibrium theory for interpreting nonequilibrium experiments. Am. J. Phys. 2006, 74, 683688. [7] Astumian, R. D.; Bier., M. Mechanochemical coupling of the motion of molecular motors to ATP hydrolysis, Biophys. Jour. 1996, 70, 637653. [8] Carter, N. J.; Cross, R. A. Mechanics of the kinesin step. Nature 2005, 435, 308312.

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