pubs.acs.org/NanoLett
Torque-Induced Slip of the Rotary Motor F1-ATPase Akilan Palanisami*,† and Tetsuaki Okamoto‡ Department of Physics, Faculty of Science and Engineering, Waseda University, Okubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan ABSTRACT F1-ATPase plays an essential role in cellular metabolism by linking rotational motion to ATP hydrolysis/synthesis. We measure the torque profile of F1 in both ATP hydrolysis and synthesis directions using a novel magnetic nanorod assay. F1 is found to decouple ATP synthesis from rotary motion at a surprisingly low torque. This low-torque slip mechanism protects the enzyme from excessive load and may play a broader biological role by reducing the production of reactive oxygen species. KEYWORDS Molecular motor, magnetism, torque, bioenergetics
F
nonconservative force, complicating comparisons with thermodynamic free energy.10 Furthermore, due to insufficient spatiotemporal resolution, these drag torque techniques (and variants)11,12 have been unable to resolve any significant variation of torque in connection with ATP binding or product release. This is important because if the torque is not constant with angle, a loaded F1 motor could stall at a torque minimum, reducing overall efficiency. Finally, this technique does not allow the controlled application of torque to F1; thus the torque behavior in the ATP synthesis direction is completely unstudied. Probing for this behavior on the single molecule level is nontrivial, requiring angstrom scale control of the F1 rotor with simultaneous measurement/application of torque. The principle obstacle to angstrom scale resolution is in maintaining the required microscope stability. Fortunately, an alternative path exists in this system, as angular motion instead of translational motion is a valid measure. By amplifying the rotational motion by means of a long rotating lever, issues of microscope stability can be obviated to achieve angstrom-precision control. Here, we expanded upon this notion by attaching a nanofabricated magnetic rod to the F1 rotor. This rod could be controlled via the magnetic field (H) from an electromagnet, allowing the precise application of torque to the F1 rotor in a conservative manner. To measure the torque, F1 was anchored to a glass surface, and a 0.29 µm diameter plastic bead bound to the γ-subunit rotor (Figure 1). A ∼1 µm long, ∼150 nm diameter14 gold-capped ferromagnetic nickel rod was then attached to the bead at 45° relative to the surface normal with the aid of H. When H is removed and ATP is added, the rod/ bead unit rotated together with the γ-rotor (F1 operates in reverse by hydrolyzing ATP and generating rotary motion) providing a convenient check of the F1 activity. The angle of the rod in the surface plane (θROD) was observed using brightfield optical microscopy. In practice, a rotating rod was found at |H| ) 0, and then |H| increased until rotation
OF1
ATP synthase, a critical component of cellular metabolism, uses proton motive force (pmf; the potential difference on opposing sides of a membrane) to generate ATP, the energy currency of the cell.1 The FO portion transduces the pmf into rotary motion, which is used to synthesize ATP by the F1 component. F1 can also act as a nanoscale (∼7 nm) rotary motor by hydrolyzing ATP to produce rotary motion with high efficiency. This dual motor/generator ability of F1 has generated considerable interest in nanoscale applications,2-4 as well as because of its metabolic implications (defects in this enzyme are typically fatal). In mitochondrial metabolism, high pmf enhances the production of reactive oxygen species (ROS), which leads to mitochondrial damage and is hypothesized to be a primary factor in aging and age-related diseases.5 Mitochondria also have a substantial proton leak, which reduces pmf and ROS production.6 Enhancing the proton leak has been shown (in some cases) to increase lifespan.7 However, the natural origins of mitochondrial proton leak have proven elusive.8 F1 may be a contributor to this leak if F1 slips during ATP synthesis (rotary energy is provided, but no ATP is produced). Although F1 is commonly taken to be nearly perfectly coupled (i.e., ATP is produced with high efficiency at all times), the conditions for F1 slip have not been determined at the single molecule level. Thus, further understanding and application of F1 hinges on elucidation of its torque behavior. In a typical hydrolysis rotation assay,9 a relatively large probe is attached to the F1 rotor, and from the speed of probe rotation the drag torque can be estimated. However, viscous drag is a fundamentally
* To whom correspondence should be addressed. E-mail:
[email protected]. † Present address: Department of Physics and Texas Center for Superconductivity, University of Houston, Houston, TX 77204. ‡ Present address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Received for review: 07/11/2010 Published on Web: 09/01/2010
© 2010 American Chemical Society
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DOI: 10.1021/nl102414d | Nano Lett. 2010, 10, 4146–4149
FIGURE 1. The F1 (r3β3γ) is attached to the glass via β (purple). Only γ (dark green) and the attached bead and rod rotate. (a) Side view. H traces out a cone over time (pink). The rod follows H along the cone. Not to scale. (b) Top view. θ increases counter-clockwise and is measured from the y-axis. (c) Sequential images taken at 1.33 s intervals with H rotating in the hydrolysis direction at 0.04 Hz. Red arrows mark jumps in torque. Individual frame width ) 2.48 µm. Crystal structure from Bowler et al.13
stopped. During torque measurement, H swept out a cone over time with the H component perpendicular to the plane (H⊥) fixed and the H component in the plane (H|) rotated. We chose |H⊥|)|H|| to maintain the rod’s 45° orientation relative to the surface. Because the H magnitude and angle (θH) in the surface plane is known, the rotary torque applied by the electromagnet15 (τEM) could be measured
τEM ∝ |H|| |sin(θDIFF)
(1)
with θDIFF ) θROD - θH. We define θ to be increasing and the sign of the τEM to be positive under ATP hydrolysis driven rotation. When the H| field was rotated in the hydrolysis direction, the resulting sin(θDIFF) versus θH plot displayed 3 fold symmetry (Figure 2a and Movie S1). As F1 is composed of a rotor piercing an “orange” containing three symmetrically placed “slices”, each containing an ATP binding pocket,13 the 3-fold symmetry of the torque profile is strong evidence that the assay is indeed probing the torque behavior of F1. Furthermore, calibration of the torque via thermal fluctuations (see Supporting Information) gave a time averaged hydrolysis τEM (Tables 1 and Supporting Information S1) consistent with previous measurements of F1 ATPase torque (∼40 pN nm).9,11,12,16,17 To further validate the magnetic torque assay, we investigated the ∼35° jumps, which are indicative of a sudden change in the torque (marked by blue arrows in Figure 2a,b) and are a previously unknown (but anticipated11) feature of the F1 hydrolysis torque profile. To elucidate the jump origin with respect to the chemical reaction scheme, a set of experiments were performed in a buffer containing an ATP regeneration system at a relatively low 500 nM ATP concentration (regeneration buffer). In the absence of an external magnetic field, the rods rotated in a stepwise fashion with three waiting states separated by 120° (Figure 2c). These pauses in rotation have been previously identified17 © 2010 American Chemical Society
FIGURE 2. (a) Torque meaurement taken with a 2 mHz rotating H in a 500 nM ATP regeneration buffer over a single hydrolysis rotation (150 000 frames of the CCD camera) plotted versus θH. Much of the noise is due to Brownian fluctuations; however, a large torque jump occurring with 3-fold symmetry is evident (marked by blue arrows). The large fluctuation at 130° breaks the symmetry, apparently a molecular “hiccup” (perhaps due to thermal fluctuations inducing a premature ATP binding/unbinding event). (b) The same data plotted versus θROD. Dots: raw data. Black line: 100 pt. running average. (c) The angle histogram of the same system released from the electromagnet. Peaks indicate ATP waiting states. 4147
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TABLE 1. Average Regeneration Buffer τEMa molecule no.
HYD (pN nm)
SYN (pN nm)
1 2 3 4 5 mean
32 ( 1 35 ( 2 49 ( 3 50 ( 3 low M Rod 42 ( 9
40 ( 1 42 ( 2 68 ( 5 60 ( 1 52 ( 14
a
Results from five different molecules studied in regeneration buffer. The average torque ((s.d.) over a single revolution was measured by executing 3 or 6 revolutions in the hydrolysis direction (HYD) or synthesis direction (SYN), excluding ADP inhibition events (see Figure S1 and related discussion in Supporting Information). θH was rotated at 0.04 Hz during measurement. In one case labeled “low M rod”, slow hydrolysis rotation was possible, but synthesis rotation had several slips, due to the weakness of rod magnetization. Torque calibration of this system was not completed due to chamber failure during the experiment. SYN/HYD for this system could be estimated (Figure 3b).
as ATP waiting states where ATP in the media binds to F1. Torque measurements done on the same system clearly connected the beginning of the torque increase to the ATP binding angle in all five systems studied under these conditions, indicating the magnetic torque assay as a sensitive probe of substrate binding induced conformation change. Having validated the magnetic torque assay using the ATPase behavior of F1, we investigated the F1 torque behavior under synthesis direction γ rotation. For F1 to synthesize ATP under regeneration buffer conditions, a minimum average torque of 73 pN nm is energetically required (see Supporting Information). However, for the majority of rotations, the mechanical work is not large enough to synthesize ATP (Table 1 and Figure 3a). ATP is synthesized from ADP and phosphate. Because the regeneration buffer scavenges ADP, F1 is being forced to rotate without ADP. Thus torqueinduced slip has been attained. The ADP dependence of the slip was further confirmed by varying the buffer condition (Figure S3 and Supporting Information). We note the slip is completely reversible (in contrast to another type of slip found in the fully assembled FOF1 holoenzyme; see Supporting Information). After all experiments, F1 was released from the electromagnet and found to perform ordinary hydrolysis rotation. To reduce the effect of torque calibration fluctuation, we normalized the average synthesis direction torque (SYN) with the average hydrolysis direction torque (HYD) for each molecule individually (Figure 3b). This reveals a collapse of the data at a normalized value of ∼1.25 and allows straightforward comparison of the average torque during slip with the average torque required for ATP synthesis. Under physiological conditions (∆GATP = 12 kcal/mol1), F1 requires a minimum average torque of 40 pN nm for ATP synthesis. The SYN is roughly 130% of this value, that is, 52 pN nm (Table 1) or 1.24 in the normalized units of Figure 3b. Surprisingly, mitochondrial proton leak also displays very similar behavior. When mitochondrial pmf is increased to 30% above the pmf of ATP production, a dramatic increase © 2010 American Chemical Society
FIGURE 3. Synthesis direction breakdown in regeneration buffer. (a) SYN is the average τEM over one rotation in the synthesis direction. A minimum SYN of 73 pN nm is required for 3 ATP per rotation synthesis (solid blue line). Some points are offset for clarity. θH was rotated at 0.04 Hz during measurement, and 7500 frames were taken with the CCD camera per rotation. Of 21 attempted rotations, 3 failures due to trap weakness and 1 anomalous rotation are not shown (see Figure S2 and discussion in Supporting Information). (b) The same data normalized by the average hydrolysis torque (HYD) for each molecule individually (taken from Table 1). The s.d. for each circular datum varied from 0.05-0.06 (not shown for clarity). The brown line indicates 52 pN nm, which is 130% of the minimum average torque required for ATP synthesis at ∆GATP ) 12 kcal/mol. To generate the normalized value, 52 pN nm is divided by the mean HYD taken from Table 1 (s.d. of brown line due to mean HYD uncertainty is indicated by the shaded region). Molecule 5 had an exceptionally weak rod magnetization, resulting in many slipped rotations. In this case, SYN/HYD was calculated from a 120° region with no magnetic interaction slips. Because of chamber failure, molecule 5 could not be torque calibrated, preventing inclusion in (a).
in mitochondrial proton leak is observed1 and suggests a connection between F1 and mitochondrial uncoupling. This is also consistent with the decrease in mitochondrial proton leak found after inhibiting ATP synthase in some prior experiments.18-21 That the F1 portion of ATP synthase may be a source of leak could not be directly investigated in these experiments due to the complexity of the mitochondrial system. These results suggest that F1 could have an additional biological role as a kind of “voltage release valve”. In this hypothesis, as the pmf increases (enhancing ROS production), γ will be increasingly likely to rotate even if ADP or 4148
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this paper published ASAP September 1, 2010. The correct version published on September 17, 2010.
phosphate has not yet bound to F1, thereby dissipating some of the pmf. Although this behavior would waste the energy stored in the pmf, this loss would be more than offset by the reduction in ROS, which degrade mitochondrial DNA and protein. In terms of nanoscale engineering, this slip mechanism creates an additional degree of robustness against load-shocks to the system. Instead of breaking, F1 can merely slip until the shock has been dissipated. The somewhat rough torque versus angle profile (Figure 2) may be problematic in some nanoengineering applications; under a varying load, F1 may get stuck at a low torque position. The solution adopted by nature has been to use an elastic coupling between the F1 and FO portions.22 This smoothes out torque fluctuations and can increase the overall efficiency of the system. In principle, such an element can be custom-engineered into the F1 rotor. We note the magnetic torque technique described here brings an added dimension to the field of molecular manipulation; AFM and optical tweezers can stretch molecules with extraordinary precision but cannot easily control motion between neighboring domains on the same protein. The magnetic nanorod assay permits this by applying torque on neighboring domains, allowing angstrom-precision control of their relative motion (kind of a mechanical analog to FRET). This method can be easily adapted for use on other enzymes.
Supporting Information Available. Detailed experimental procedures, expanded discussion, additional data, and a movie clip. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES AND NOTES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)
Acknowledgment. We thank the Waseda University machine shop for assistance in microscope design; Electroplating Engineers of Japan Ltd. for the gift of electroplating solutions; M. Yoshida for E. coli. strains for F1 expression; Y. Oono and B. Spring for comments on the manuscript; and members of the Kinosita laboratory for assistance. We especially thank K. Kinosita for his generous advice, encouragement, and support throughout this project. A.P. was supported by a JSPS postdoctoral fellowship. Support was also provided by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
(15) (16) (17) (18) (19) (20) (21)
Note Added after ASAP Publication. Equation 3 in the Supporting Information has been modified in the version of
© 2010 American Chemical Society
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