ΔμH+ Dependency of Proton Translocation by Bacteriorhodopsin and

On the other hand, proton transfer from Asp96 to the Schiff base during the decay of the M intermediate can account for the large effect of Δψ on th...
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J. Phys. Chem. 1996, 100, 19687-19691

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∆µH+ Dependency of Proton Translocation by Bacteriorhodopsin and a Stochastic Energization-Relaxation Channel Model Eiro Muneyuki,*,† Mineo Ikematsu,‡ and Masasuke Yoshida† Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan, and Tsukuba Research Center, Sanyo Electric Co., Ltd., Tsukuba, Ibaraki 305, Japan ReceiVed: May 23, 1996; In Final Form: September 17, 1996X

The effect of a pH gradient (∆pH) and a membrane potential difference (∆ψ) on the rate of proton translocation by bacteriorhodopsin was examined. Contrary to the “proton well” hypothesis, variation of ∆ψ exerted a considerably larger effect on the rate of proton translocation than the energetically equivalent magnitude of ∆pH (maximum ∆pH examined was about 2, while the absolute pH value was 5-9.) These apparently puzzling features are, however, consistent with the structural data, particularly in view of an asymmetric environment provided by the key amino acid residues with different pKa values. The relatively small effect of ∆pH is explained in terms of the proton uptake residue, Asp96, and the proton-ejecting residue, Asp85, whose pKa values are known to be about 10 and 3 in the ground state, respectively. On the other hand, proton transfer from Asp96 to the Schiff base during the decay of the M intermediate can account for the large effect of ∆ψ on the rate of proton translocation. With these experimental data and explanations in mind, we further propose a simplified stochastic model for proton pumping where an asymmetric environment, which in turn provides an asymmetric potential field for protons, plays an essential role for vectorial proton translocation. A simple numerical simulation could qualitatively reproduce the experimental data. These results suggest that some common principle may exist in the mechanisms of ion pumps and molecular motors, and it may be applied in development of an artificial ion pump molecule.

Since the proposal of the chemiosmotic theory,1 the significance of the conversion of vectorial osmotic energy and scalar (photo)chemical energy has been established. However, a mechanism for how a protein molecule can mediate this interconversion remains to be elucidated. For example, the question of how a H+ pump protein responds to energetically equivalent proton concentration gradients (∆pH) and electric membrane potentials (∆ψ) has not been clearly answered. Actually some data indicate they are both thermodynamically and kinetically equivalent,2-4 whereas some other data indicate there was some difference between them.5 However, a clear conclusion could not be drawn due to the lack of a reliable technique to control the ∆pH and ∆ψ imposed on the operating ion pumps. The artificial planar bilayer system is ideal in that it enables us to control independently ∆pH and ∆ψ on the electrogenic ion pumps;6-8 however, incorporation of ion pumps into planar bilayers was not easy, and this system has been applied mostly for the research on ion channels. Recently, we have developed a method to incorporate a lightdriven ion pump, bacteriorhodopsin (bR), directly into planar bilayer membranes.9 Judging from the effects of a protonophore, FCCP, and a side-specific inhibitor, LaCl3, it was concluded that most of the steady state photoelectric current was generated by bacteriorhodopsin molecules which were reconstituted unidirectionally in a transmembranous manner.9 By using this system, we have investigated the effect of ∆pH and ∆ψ on the rate of light-driven proton translocation by bR. It was clearly shown that the variation of ∆ψ exerted a considerably larger effect on the rate of proton translocation than the energetically equivalent ∆pH (maximum pH examined was about 2, while the absolute pH value was 5-9). The small * To whom correspondence should be addressed. Tel: (Japan)-45-9245232. Fax: (Japan)-45-924-5277. E-mail: [email protected]. † Tokyo Institute of Technology. ‡ Sanyo Electric Co., Ltd. X Abstract published in AdVance ACS Abstracts, November 1, 1996.

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effect of ∆pH was explained in terms of the proton uptake residue, Asp96, and the proton-ejecting residue, Asp85, whose pKa values are known to be about 10 and 3 in the ground state, respectively. On the other hand, proton transfer from Asp96 to the Schiff base during the decay of the M intermediate can be a responsible step for the large effect of ∆ψ on the rate of proton translocation. With the known structure of bR, a simple model based on the in-line positioning of a key proton uptake residue with a high pKa, an intermediate residue with a variable pKa, and a proton-ejecting residue with a low pKa is proposed. This model assumes only a simple stochastic behavior for the pKa change of the intermediate residue. A Monte-Carlo simulation based on this model could qualitatively reproduce the experimental data. Furthermore, we suggest a similarity of our present model to the thermal ratchet mechanism proposed for molecular motors. The simple principle for proton pumping described in this paper may be applied in the development of an artificial ion pump molecule. Materials and Methods Bacteriorhodopsin (bR) was incorporated into planar bilayer membranes by a previously reported method,9 and the proton translocation by bacteriorhodopsin was directly measured as photoelectric current. The effect of a protonophore, FCCP, and a side-specific inhibitor, LaCl3, indicated that most of the steady state photoelectric current was generated by bacteriorhodopsin molecules which were reconstituted unidirectionally in a transmembranous manner.9 The program for the computer simulation was written in C language which runs on an NEC PC-9800 series personal computer or its compatible machines. The rate constants for the simulation were set as follows (see also Figure 3). The on constants of protons to site A (k+1) and site C (k-4) were estimated as 1011 and 1010 M-1 s-1, which are reasonable values © 1996 American Chemical Society

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Muneyuki et al.

as the rate constants for protonation. In order to satisfy the pKa of 11 and 3 for site A and site C, the off constants from site A (k-1) and C (k+4) are 100 and 107 s-1, respectively. As we assume that the proton transfer from site A to site B under a deenergized state corresponds to the decay of the M intermediate of bR, the rate constant for the proton transfer (k+2) should be 102 s-1. Combined with the pKa for site B of 13, which corresponds to the pKa of the Schiff base in bR in the deenergized state, the rate constant for proton transfer from site B to site A (k-2) is 100 s-1. As for the k+3 and k-3, we arbitrarily assign 10-8 and 102 s-1, respectively. For the energized state, there is less rationale to assign values to the rate constants. As the pKa of site B is assumed to be 2 in the energized state, the ratio of k+3 to k-3 is 10. Keeping k-3 the same as in the deenergized state (102 s-1), k+3 should be 103 s-1. In order to make k+3 and k-2 symmetrical, k-2 is also assumed to be 103 s-1, and hence k+2 is 10-6 s-1. To satisfy microscopic reversibility, k+1k+2k+3k+4 is equal to k-1k-2k-3k-4 for the energized and deenergized states, respectively. The electric distances between sites A and B and between sites B and C were assumed to be 0.0 and 1.0, for simplicity. The results of single turnover experiments revealed that they are actually about 0.75 and 0.25.10,11 The voltage dependence of individual rate constants was assumed to follow Eyring’s rate theory.12 Note that the assumptions on the rate constants are made in order to carry out a simple numerical simulation and should not be regarded as a precise modeling of the bR photocycle; see also Results and Discussion. Simulation was carried out by a MonteCarlo method. The probability for the energization or relaxation was set to 0.001 for each step. One step of calculation corresponded to 10 µs. Results and Discussion The light-driven proton translocation by bR was directly measured as photoelectric current after reconstitution into planar bilayer membranes. The effect of a protonic electrochemical potential (∆µH+) on the rate of proton translocation was examined by changing ∆pH and ∆ψ independently. In Figure 1A, the pH of one side was decreased to 5 by adding small aliquots of acid while the pH of the other side was kept at 7. In Figure 1B, the pH of one side was increased to 9 by adding small aliquots of base while the pH of the other side was kept at 7. In both cases, it is clear that the photoelectric current generated by bR upon illumination was rather insensitive to ∆pH under the experimental conditions. In Figure 1C, ∆ψ was varied while the pH values of both sides of the membrane were kept at 7. Clearly, the photoelectric current was affected by ∆ψ to a larger extent than by ∆pH. At around -90 mV, the photoelectric current virtually disappeared, which agrees with the ∆ψ dependency reported by another group.13 The independent effects of ∆ψ and ∆pH on the rate of proton translocation by bR under the same conditions were examined for the first time here. These results are apparently in contrast to the “proton well” hypothesis;14 however, they are interpreted to mean the following situations. The apparent absence of ∆pH dependency on the rate of proton translocation suggests that (1) the affinity for protons of the proton-uptaking site at the cytoplasmic side is very high in a proton-binding step. The site should have a high pKa in order to bind a proton at pH 5-9; (2) the affinity for protons of the proton-releasing site at the extracellular side is very low in a proton-releasing step. This site should have a low pKa in order to release a proton at pH 5-9. In addition, binding and release of the transported protons are not rate limiting.

Figure 1. Relationships between the ∆µH+ and the rate of proton translocation. Different symbols stand for independent experiments. The magnitude of the electric current was normalized by the value at zero ∆µH+. (A) ∆pH varied. The pH of one side of the membrane was decreased by adding aliquots of dilute acid, while the pH of the other side was kept at 7. Then, the pH of the acidic medium was returned to 7 by perfusion with a neutral buffer, and the dilute acid was added to the other side to make a reversed pH gradient. The membrane potential (∆ψ) was kept at 0 mV by an external feedback electric circuit (voltage clamp). (B) ∆pH varied. The pH of one side of the membrane was increased by adding aliquots of dilute base, while the pH of the other side was kept at 7. The pH of the other side was changed after perfusion as in A. ∆ψ was kept at 0 mV as in A. (C) ∆ψ varied. Membrane potential was varied by external electric circuits under voltage clamp conditions. The pH values of the medium of both sides of the membrane were kept at 7.

On the other hand, the relatively steep ∆ψ dependency suggests that the major rate-limiting step is voltage dependent. These properties fit well with the known structure and the molecular events during the photocycle of bR. Asp96 of bR has been assigned as the key residue for uptaking protons from the cytoplasmic side and proved to have a very high pKa (>10) in the ground state.15 This means that Asp96 can capture a proton irrespective of the pH (5) of the extracellular side. Thus, the rate of proton translocation shows little dependency on ∆pH when the pH value is between 5 and 9. It is known that one of the major rate-limiting steps in the photocycle of bR is the decay of the M intermediate, which involves proton transfer from Asp96 to the Schiff base.17 The pKa of the Schiff base is estimated to be 13 in the ground state,

Proton Translocation by bR

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Figure 2. Simplified mechanism for proton pumping from high-pH side to low-pH side. See text for the explanation of the model.

and it changes during the photocycle.18 Asp96 and the Schiff base are about 12 Å apart from each other,19 and 60% of the electric potential is generated during the decay of the M intermediate.10,11 Such a charge movement in a low-dielectric environment which does not shield the external electric field should be strongly affected by ∆ψ, and this can account for the steep ∆ψ dependency. This explanation is consistent with the observation that the lifetime of the M intermediate depends on ∆ψ rather than ∆pH.20 Thus, the macroscopic ∆pH and ∆ψ dependency of the proton translocation by bR can be well understood on the basis of the molecular mechanisms. Among them, the most important factor which determines the macroscopic feature of the proton translocation is the asymmetric environment provided by the key amino acid residues with different pKa values. Furthermore, we think that the asymmetric environment plays an essential role in transducing scalar light energy into vectorial proton movement and propose a simplified mechanism for proton pumping which is schematically shown in Figure 2. The model assumes three rules. (i) There are three binding sites for protons, and protons migrate among these sites. (ii) The binding site responsible for proton uptake from the high-pH side (site A) has a high pKa. The binding site for ejecting protons to the low-pH side (site C) has a low pKa. The binding site between site A and site C (site B) has a variable pKa. At the ground state, the pKa of this site is the highest of the three, and when energized, it becomes the lowest. (iii) The lifetime of the energized, low-pKa state of site B is sufficiently long to prevent transfer of a proton from site C to site B. Given these assumptions, the vectorial proton translocation can be achieved as follows. In the ground, deenergized state, both sites A and B are protonated whereas site C is deprotonated in accordance with the pKa values of these sites (Figure 2A). When energized, the pKa of site B is drastically lowered, favoring release of a proton from site B (Figure 2B).

Figure 3. Schematic presentation of the stochastic energizationrelaxation channel model. This figure represents hypothetical potential surfaces for proton translocation. Left aqueous phase and right aqueous phase correspond to the cytoplasm and extracellular medium for bR. Very roughly speaking, sites A, B, and C correspond to Asp96, Schiff base, and Asp85 in bR, respectively. Stochastic fluctuations between the energized and relaxed states (energization and relaxation) cause the decrease and increase of the pKa of site B shown in parts B and E of Figure 2, respectively. The protons diffuse on the potential surfaces according to the rate constants of the energized and relaxed states. Medium pH affects rate constants of k+1 and k-4, whereas the membrane potential affects the other rate constants according to the electric distance between proton-binding sites.

The proton released from site B can bind only to site C because site A is already occupied by another proton (Figure 2C). The proton bound to site C has a high tendency to dissociate because of the low pKa of site C. On the other hand, the lifetime of the energized, very low pKa state of site B is assumed to be long enough to prevent a proton transfer from site C, and the proton released from site C diffuses into the right water phase (low-pH side) without returning to site B (Figure 2D). When site B relaxes to the highest pKa state, only site A can provide a proton to site B, and thus a proton is transferred from site A to site B (Figure 2E). Finally, site A takes up a proton from the left water phase (high-pH side) which completes the cycle (Figure 2A). In this model, coupling of the energization and proton translocation is essentially stochastic and loose. The transition of site B from the highest pKa state to the lowest pKa state (Figure 2B) and its relaxation (Figure 2E) are equivalent to the stochastic fluctuation of the asymmetric potential field for protons as shown in Figure 3. This potential profile is similar to that of multiion channels.12 According to this energizationrelaxation channel model, a vectorial movement of protons is induced by the random fluctuations of an asymmetric potential

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Figure 4. Simulated ∆µH+ dependency of the proton translocation based on the stochastic energization-relaxation channel model (Figure 3). Simulation was carried out by a Monte-Carlo method. For the details of the conditions, see Materials and Methods. A, B, C correspond to A, B, C in Figure 1, respectively.

field provided by the key proton-binding sites (amino acid residues) with different pKa values shown in Figure 3. An important point of this model is that this model satisfies the microscopic reversibility in both the energized and deenergized states. That is, the products of the rates of all forward and backward reactions in proton transfer are equal in the energized and deenergized states, respectively. It seems necessary to satisfy the microscopic reversibility for all the defined states because if the microscopic reversibility is betrayed at any state, the state works as a perpetual motion machine. Thus, not only the energization but also the relaxation is essential for a continuous proton translocation. On the basis of this simplified model, we carried out a Monte-Carlo simulation and examined the effects of ∆pH and ∆ψ. In spite of the extensive simplification, the simulation could qualitatively reproduce the experimental results (Figure 4). Of course, this model is extensively simplified to highlight the key principle, and it differs in many points from the actual bR mechanism.21 The most obvious difference is that when we assume that site B and site C correspond to the Schiff base and Asp85 of bR, Asp85 should deprotonate immediately after receiving a proton from the Schiff base; however, this is not the case.22 Actually, the proton-ejecting mechanism in bR involves Asp85 and Glu204, of which Asp85 participates in the internal proton transfers and Glu204 releases protons to the extracellular side at physiological pH.23 The pKa of Asp85 increases during the photocycle,24 and Asp85 and Glu204 interact each other to change their pKa values to facilitate proton

Muneyuki et al. release.25 It was proposed that at pH > 5.8, the transported proton is released from XH, which is most probably Glu204,23 and at pH < 5.8, the proton is released from Asp85 directly to the extracellular side.26 In this case, site B in the present model cannot simply be assumed to correspond to Asp85 and the simple model presented here fails to explain the order of proton binding and release at lower pH. More intermediate states and proton-binding sites should be involved to fit the model precisely to the actual bR. In the actual bR mechanism, some water molecules between the Schiff base and Asp85 may play a critical role.27 In the actual bR, the pKa of Asp85 increases during the photocycle and it is not necessary for the pKa of the Schiff base to decrease to as low as 2 assumed in our model. The pKa of the Schiff base was shown to decrease to near 8 in D96N mutant,28 and ∆pKa of Asp85 and the Schiff base becomes nearzero during the proton transfer from the Schiff base to Asp85. The assumption that site A and site C have a constant pKa throughout the cycle is made for simplicity to allow a simple numerical simulation. Recently, it was reported that deprotonation of Asp96, which roughly corresponds to site A in our model, takes place in spite of the absence of the unprotonated Schiff base of the M intermediate in D212N mutant, strongly suggesting a decrease in the pKa of Asp96 during the photocycle.29 Due to these many differences, the model presented here should not be regarded as a precise description of bR itself; however, we believe that our model contains some essential principle in active transport which includes the following: (1) There must be at least two different states which provide asymmetric potential fields for the transported protons, and the vectorial transport is induced by the transition between these states. (2) At each state, the microscopic reversibility should be satisfied in the proton transport reactions. In other words, the proton transfer can occur stochastically satisfying the potential differences between each proton-binding site and the aqueous phase. (3) The transitions between the different states (energization and/or relaxation) may occur stochastically independent of the proton transfers, but the transition should not be too fast. Probably, bR has gained many additional pertinent features during evolutional processes to improve the efficiency of energy transduction by having many proton-binding sites and complex potential field for the transported protons in each of the photointermediates. In this sense, our model might be regarded as a model for the ancient ancestor proton pump of bR or as a starting point in understanding the properties of proton translocation by a stochastic model. Obviously, our model should also evolve to contain more intermediate states based on the precise knowledge of the pKa values of key residues in each photointermediate and rate constants of the transitions to describe the real bR. This would make the model more complex, but the principle in the present model remains the same. Although our model does not include any conformational change of the protein explicitly, the conformational change is included as the change of the proton affinity and accessibility between site A and site B and between site B and site C. This is envisaged as the change of rate constants k+/-2 and k+/-3 in simulation and the change of potential profile schematically shown in Figure 3. Thus, our model does not contradict the alternative protein conformation model originally proposed as a general mechanism for ion pumps30,31 and which assumes conformation E and conformation C for bR.21,32 In some models, it seems that breaking of microscopic reversibility is assumed in one conformational state;33 however, this is excluded in our model. Therefore if a conformational state which allows

Proton Translocation by bR breaking of microscopic reversibility or not is a very important problem to be elucidated. In addition, the stochastic feature may give our model flexibility to explain many seemingly complex experimental results such as the inversion of proton translocation by some mutant bRs,34 the switching of the proton-releasing group at different pH values,26 and the appearance of the MN state35 in D96N mutant at high pH. Finally, we would like to point out the similarity of the present model for proton pumping to the fluctuation-driven ratchet mechanisms which are regarded as models for the contraction of muscle fibers or for the transport of macromolecules along microtubles.36-41 According to the fluctuation-driven ratchet mechanisms, symmetry breaking and substantially long time correlation are essential to induce directed motion from random noise. In the present model, the symmetry breaking is provided by the strategically placed key proton-binding sites (amino acid residues) with different pKa values within the ion pump molecule, and slow protein conformational change gives a long time correlation. In order to extract vectorial motion from noise, the simple ratchet explanation may appear to contradict the second law of thermodynamics or to require the help of Maxwell’s demon;42 however, this is not the case in our present model. In the energization step, the energy input is clearly defined as to elevate the potential of the proton bound to site B (Figure 3). Thus, in spite of the differences in the actual molecular events between bR and motor proteins, we suspect that some similar principles play essential roles in these biological energy transduction systems. Furthermore, this simple principle may be applied to construct an artificial ion pump in the future. References and Notes (1) Mitchell, P. Nature 1961, 191, 144-148. (2) Thayer, W. S.; Hinkle, P. C. J. Biol. Chem. 1975, 250, 53365342. (3) Slooten, L.; Vandenbranden, S. Biochim. Biophys. Acta 1989, 976, 150-160. (4) Gra¨ber, P. Bioelectrochemistry III; Plenum Press: New York, 1990; pp 277-309. (5) Schmidt, G.; Gra¨ber, P. Biochim. Biophys. Acta 1987, 890, 392394. (6) Montal, M. Annu. ReV. Biophys. Bioeng. 1976, 5, 119-175. (7) Darszon, A. Methods Enzymol. 1986, 127, 486-502. (8) Bamberg, E.; Dencher, N. A.; Fahr, A.; Lindau, M.; Heyn, M. P. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 7502-7506. (9) Muneyuki, E.; Ikematsu, M.; Iseki, M.; Sugiyama, Y.; Mizukami, A.; Ohno, K.; Yoshida, M.; Hirata, H. Biochim. Biophys. Acta, 1993, 1183, 171-179.

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