Direct observation of stepped proteolipid ring rotation in E. coli FoF1-ATP synthase
2010; Springer Nature; Volume: 29; Issue: 23 Linguagem: Inglês
10.1038/emboj.2010.259
ISSN1460-2075
AutoresRobert Ishmukhametov, Tassilo Hornung, David Spetzler, Wayne D. Frasch,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoArticle29 October 2010free access Direct observation of stepped proteolipid ring rotation in E. coli FoF1-ATP synthase Robert Ishmukhametov Robert Ishmukhametov Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Tassilo Hornung Tassilo Hornung Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author David Spetzler David Spetzler Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Wayne D Frasch Corresponding Author Wayne D Frasch Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Robert Ishmukhametov Robert Ishmukhametov Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Tassilo Hornung Tassilo Hornung Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author David Spetzler David Spetzler Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Wayne D Frasch Corresponding Author Wayne D Frasch Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Author Information Robert Ishmukhametov1,‡, Tassilo Hornung1,‡, David Spetzler1,‡ and Wayne D Frasch 1 1Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ, USA ‡These authors contributed equally to this work *Corresponding author. School of Life Sciences, Arizona State University, Box 874501, Tempe, AZ 85287 USA. Tel.: +1 480 965 8663; Fax: +1 480 965 6899; E-mail: [email protected] The EMBO Journal (2010)29:3911-3923https://doi.org/10.1038/emboj.2010.259 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the FoF1-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in Fo has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between Fo subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli FoF1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c-ring to a limit of ∼36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the Fo motor where c-ring rotational diffusion is limited to 36°. Introduction The FoF1-ATP synthase is composed of two opposed rotary molecular motors connected by a common axle of γε-subunits (Stock et al, 1999). The integral membrane Fo motor, which has a subunit stoichiometry of ab2c10 in Escherichia coli (Jiang et al, 2001), uses the electrochemical potential-driven flux of protons across a membrane (proton-motive force (PMF)) to drive clockwise rotation of the ring of 10 c-subunits as viewed from the periplasm (Börsch et al, 2002). The c-ring is docked to the γε-subunits that extend into the hexameric ring of α- and β-subunits in the F1 peripheral membrane motor. Rotation of this axle drives conformational changes in each of the three catalytic αβ heterodimers resulting in ATP synthesis (Boyer, 1997). The F1 motor can also hydrolyze ATP resulting in counterclockwise γε-subunit rotation and proton translocation via Fo (Börsch et al, 2002). When solubilized away from Fo and the membrane, E. coli F1-ATPase-driven rotation at saturating ATP concentrations occurs in three 120° power strokes (Sabbert et al, 1996; Noji et al, 1999; Spetzler et al, 2006), separated by 8.3 ms dwells comparable to the turnover time of the rate-limiting step of ATP hydrolysis (Spetzler et al, 2006; Hornung et al, 2008). In the absence of drag on the F1 motor, the velocity of the power stroke is ∼0.5° μs−1 (Spetzler et al, 2006). In vivo, FoF1 uses the PMF across the membrane to maintain the [ATP]/[ADP][Pi] ratio (Q) far from equilibrium so that the high-ATP concentration provides an energy source to drive other cellular processes. Energetically, this means that at steady state, cellular PMF≅2.3RTlogQ. In other words, the driving force of the Fo motor (PMF) is in equilibrium with the driving force of the F1 motor (logQ). In E. coli, the cytoplasm typically contains 3 mM ATP, 0.4 mM ADP, and 6 mM Pi such that logQ≅0.1 (Weber and Senior, 1997). The maximum reported rate of E. coli FoF1 ATP synthesis (Senior et al, 2002) is about 100 s−1 (10 ms ATP−1), although rates of 27 s−1 (37 ms ATP−1) are more common with E. coli FoF1 in proteolipisomes (Fischer et al, 1994). Proton translocation can occur at faster rates when powered by ATP hydrolysis (Feniouk and Junge, 2008), or by membrane potentials imposed on E. coli Fo-embedded membranes after removal of F1 (Franklin et al, 2004; Wiedenmann et al, 2008). In the absence of F1 there is no indication that the proton translocation rate of Fo saturates at high-driving force (Feniouk and Junge, 2008), which suggests that the proton translocation step is not rate limiting to the mechanism. Proton translocation across the membrane occurs in Fo when subunit-a residue aR210 deprotonates the cD61 carboxyl on each c-subunit as the c-ring rotates (Fillingame et al, 1984; Lightowlers et al, 1987; Angevine et al, 2003; Ishmukhametov et al, 2008). A Brownian ratchet mechanism has been postulated to power Fo rotation that must meet two requirements to function (Junge et al, 1997; Oster et al, 2000): first, that there are two noncolinear proton access half-channels from each side of the membrane leading to the cD61 carboxyl; and second, that rotational diffusion of the c-ring relative to subunit-a is periodically restricted in some manner. However, experimental evidence that provides a molecular basis for the latter requirement is scarce. Initial single-molecule c-ring rotation measurements of FoF1 driven by ATP hydrolysis, or by an electrochemical potential, resolved only 120° steps (Sambongi et al, 1999; Pänke et al, 2000; Börsch et al, 2002; Kaim et al, 2002; Nishio et al, 2002; Ueno et al, 2005). C-ring rotation with step sizes of 36°, 72°, 108°, and 144° occurring 48, 37, 12, and 3% of the time, respectively, have now been observed using E. coli FoF1 proteoliposomes that synthesized an ATP every 37 ms in response to a membrane potential of >200 mV (Düser et al, 2009). As the proton translocation rate of Fo does not saturate at high-driving force (Feniouk and Junge, 2008), the movement of c-subunits past subunit-a during the 72°, 108°, and 144° steps may not have caused a dwell in rotation. We now report the observation of a previously unknown interaction between Fo subunits a and c of FoF1 when ATPase-driven rotation is slowed by a viscosity-induced load. A striking feature of this interaction is that it forms a leash that limits rotation to ∼36° in a manner that can satisfy the restricted motion requirement in the Fo Brownian ratchet mechanism. As the transient dwells do not form when c-ring rotation and proton transport occur at high rates, the practical advantage of using the leash is anticipated to be under steady-state conditions in which the cellular ATP concentration is high relative to ADP and Pi. Under these conditions, when the free energy of the proton gradient approaches equilibrium with the chemical potential of ATP, the Fo motor could use this leash as part of a Brownian ratchet to bias rotation for ATP synthesis (clockwise) against an F1 motor-imposed load. Results FoF1 nanodiscs are fully assembled and retain complete activity during single-molecule measurements To stabilize the hydrophobic Fo complex, we inserted solubilized FoF1 into phospholipid bilayer nanodiscs. The particle size of nanodiscs is constrained by the membrane scaffold protein (MSP) construct MSP-1E3D1 that forms a 13-nm diameter ring of α-helices around a bilayer of phospholipid molecules, and has been shown to provide a good model for lipid bilayers (Bayburt et al, 2007). The nanodiscs are large enough to allow the incorporation of the Fo complex and a few hundred lipid molecules, yet are on the same scale as the FoF1 complex. Assembly of stable nanodisc-FoF1 complexes (n-FoF1) from MSP, lipids, and detergent solubilized FoF1 was verified by 2D electrophoresis (Figure 1). The first nondenaturing gel dimension contained one prominent band. This band contained both MSP and the FoF1 subunits when separated in the second denaturing gel dimension. The absence of other bands in the nondenaturing gel corresponding to incomplete n-FoF1 constructs suggests that the majority of proteins contain the full complement of subunits. Figure 1.Incorporation of FoF1 into nanodiscs. Two-dimensional electrophoresis gel of purified FoF1 before (left lane) and after (right lane) incorporation into nanodiscs. (A) Dimension 1: Coomassie-stained 5–15% nondenaturing gel. (B) Dimension 2: Silver-stained 15% denaturing gel separating proteins in the single-band excised from the nondenaturing gel. (C) ATPase activity of detergent solubilized FoF1 () and n-FoF1 () versus time at 25°C normalized to the initial activity of 110 and 145 s−1, respectively. Download figure Download PowerPoint We made the c2∇C mutation to a cys-free E. coli FoF1 enzyme that inserted a sulfhydryl group on each c-subunit for covalent modification with biotin maleimide, which is designated here as FoF1. Figure 1C shows the ATPase activity of n-FoF1 versus detergent solubilized FoF1 as a function of time. Detergent solubilized FoF1 lost all activity and aggregated within a few hours at room temperature. In comparison, the activity of n-FoF1 was initially higher and did not decline significantly after the preparation had been at 25° for 8 h. Modification of cD61 in the c-ring of n-FoF1 by N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity by as much as 85% (Table I), indicating that there was strong coupling between hydrolysis and proton transport. This extent of inhibition is comparable to that reported by Ueno et al (2005) for detergent solubilized FoF1 used in single-molecule rotation studies. The rapid loss of activity of detergent solubilized enzyme may explain why Ueno et al (2005) observed rotation of only 60 detergent solubilized FoF1 molecules in 840 fields of view. Table 1. Biochemical characterization of FoF1 mutants that lack transient dwells Strain kcat ATPase (s−1) kcat ATPase +DCCD (s−1) ATPase in SBP μmol ATP min−1 mg protein−1 ATPase-dependent proton pumping (% of WT) n-FoF1 (WT) 140 21 1.8 100 n-FoF1-cD62Ga 130 130 0.8 0 n-FoF1-aR210G 20 20 1.1 0 n-FoF1-a∇14 90 90 0.4 0 a cD62 is so named due to the c2∇C insert mutant used for biotinylation. Biotinylated n-FoF1 was attached to a cover slip via 6xHis-tags on the β-subunit N-terminus. Subsequent addition of avidin-coated gold nanorods then became bound to the biotins positioned on the c-ring distal from F1 (Figure 2A). Nanorods observed in Figure 2B were specifically bound to the c-ring of FoF1 on the microscope slide as n-FoF1 that lacked the c2∇C mutation failed to bind nanorods (Figure 2C). The stability of ATPase activity of the n-FoF1 complex (Figure 1C) is important due to the time required to complete single-molecule experiments. The abundance of n-FoF1 observed to rotate was at least 25% of the molecules in an average field of view that typically contained about 250 molecules (Figure 2B), which was comparable to the abundance observed using purified F1-ATPase (York et al, 2007). Figure 2.FoF1 nanodiscs (n-FoF1) in single-molecule rotation studies. (A) Microscope slide bound n-FoF1 attached via β-subunit N-terminus 6 × His tags attached to an avidin-coated 77 × 39 nm2 nanorod via a biotinylated subunit-c cys. (B, C) Microscope fields-of-view of gold nanorods (red and green dots) bound to a slide coated with n-FoF1 in which subunit-c contained (B) or lacked (C) the cys insertion mutation. Download figure Download PowerPoint High-speed rotational power stroke measurement using gold nanorods As shown in Figure 3, gold nanorod rotation results in a change in the intensity of red light scattered from the nanorod when viewed through a polarizing filter (Sönnichsen and Alivisatos, 2005; Spetzler et al, 2006). The intensity of red scattered light from a nanorod changes in a sinusoidal manner as a function of the rotary position of the nanorod relative to the plane of polarization with minimal and maximal intensities separated by 90° (Spetzler et al, 2006). Figure 3B shows the distribution of scattered red light intensities from a single-nanorod immobilized to the surface of a microscope slide as a function of the rotational position of the polarizing filter. At each position of the polarizer, the scattered light intensity was sampled 3520 times under conditions comparable to that used to measure rotation of n-FoF1 molecules. The sample number of 3520 was used because it corresponds to the average number of rotational power stroke events measured for each n-FoF1 molecule during the 50-s data acquisition period used for all measurements reported here. Figure 3.Use of nanorods to measure c-ring rotation of n-FoF1. (A) Micrographs of white light scattered from a single-gold nanorod viewed through a polarizing filter, and as a schematic showing the orientation of the nanorod to the polarizer. Measurement of rotation position is made only with the intensity of polarized red light scattered from the nanorod. (B) Histograms of the intensity of red light scattered from a single nonrotating nanorod fixed to a slide as a function of the rotational position of the polarizer. Each histogram contains 3520 measurements at each position of the polarizer obtained with the data acquisition speeds used to collect data points for c-ring rotation. The polarizer was then rotated counterclockwise by 10°, and data collection was repeated. (C) Standard error of nanorod rotational position versus degrees of rotation of the polarizer from the minimum intensity of light scattered from the nanorod as determined by Equation (1). (D) Relationship between a 120° power stroke and a 90° measured rotational transition. Theoretical plot of the intensity of scattered red light from a nanorod during one complete revolution that involves three consecutive power strokes and three consecutive catalytic dwells separated by exactly 120°. The nanorod is initially positioned almost, but not exactly perpendicular to the orientation of the polarizer such that the scattered light intensity goes through a minimum then a maximum prior to catalytic dwell 1. A transition includes the data between the minimum and maximum intensities representing 90° of the 120° of rotation for analysis. When initial alignment of the nanorod is exactly at the minimum and each of the successive power strokes is exactly 120°, the algorithm selects transitions for power strokes 1 (min to max) and 3 (max to min). Download figure Download PowerPoint The scattered light intensity from the nanorod in Figure 3B varied between maximum and minimum values of 2500 and 500. The difference between these values resulted in a dynamic range of about 2000 photons per sample, which determined the sensitivity of the measurement. This was the minimum dynamic range used to measure rotation (the average range was ∼3000 photons per sample), and thus serves as the upper limit for determining the error in the measurement of rotational position. The error in the determination of rotational position primarily results from variations in the intensity of scattered photons from the nanorod. The distribution of light intensity scattered from the nanorod was narrower at polarizer angles in which the intensity was at a minimum than that observed at the maximum. The degrees of rotation during a transition were derived from the arcsine of the fractional intensity of light scattered from the nanorod by Equation 1: where θ is degrees of rotation, and I is the fractional intensity of scattered light. Consequently, the standard error in the measurements of Figure 3B varied between about 0.02 and 0.12 degrees as the scattered light intensity varied between minimum and maximum values (Figure 3C). A saturating concentration of 1 mM Mg2+-ATP was used for all rotational measurements reported here. Under this condition, F1-ATPase-dependent power strokes occur in uninterrupted 120° rotational events separated by 8.3 ms catalytic dwells (Spetzler et al, 2006). A schematic of scattered light intensity during three consecutive power strokes (one complete revolution) is shown in Figure 3D when the nanorod was initially aligned nearly, but not exactly, perpendicular to the polarizer. As the stochastic nature of the enzyme results in a variation in the rotational position of each catalytic dwell (Yasuda et al, 2001), the alignment of the nanorod with the polarizer will show small variations during the data collection period. Consequently, the most sensitive and precise measure of rotational position during a power stroke is obtained when the nanorod rotates through the parallel and perpendicular alignment of the polarizer during a single 120° power stroke. This was measured as a change between maximum and minimum intensities of the scattered light, which corresponds to 90° of rotation, and an algorithm was used to collect these data as described previously (Spetzler et al, 2006). The time required for 90° of continuous rotation to occur is defined as the transition time (Figure 3D). If the nanorod is initially aligned perpendicular to the polarizer and the three consecutive power strokes are exactly 120° during a single revolution such that the nanorod is also perpendicular during catalytic dwell 3, the algorithm will analyse transitions from power strokes one and three. In practice, the number of consecutive power strokes analysed is randomized by the stochastic nature of the molecular motor. Due to the randomization, there is an equal probability that the 90° increments of rotation measured as transitions represents the beginning, the middle, and the end of each 120° power stroke such that the entire power stroke is sampled in the course of the ∼3520 power stroke events monitored for each molecule during the 50-s of data acquisition. Appearance of transient dwells independent of proton translocation In single-molecule studies of 320 n-FoF1 molecules (∼3520 transitions molecule−1), we observed two populations of n-FoF1 molecules based on differences in the transitions (Figure 4A, open and solid black squares) acquired during the 50-s of rotation measured for each molecule. The transitions in one population appeared nearly identical to those observed with isolated F1-ATPase-driven rotation (circles). These transitions were similar in that the power strokes of both rotated continuously for the full 90° of the transition and achieved equivalent velocities. The other population of n-FoF1 molecules took much longer to complete a 90° transition due to the appearance of transient dwells. The transient dwells were present in >90% of the power strokes of any n-FoF1 molecule in this latter population, whereas 80% for PEG400 concentrations between 15 and 35%. A similar trend was observed with n-FoF1-aR210G and n-FoF1-cD62G molecules as a function of PEG400 concentration although the total abundance was somewhat lower than that observed with n-FoF1. Figure 7.Effects of viscosity on the appearance and duration of transient dwells. (A) Abundance of n-FoF1 (Δ), n-FoF1-aR210G (), FoF1-cD62G (), and n-FoF1-a∇14 () molecules with transient dwells as a function of PEG400 concentration. (B) Transient dwell duration of n-FoF1 (), n-FoF1-aR210G (), and n-FoF1-cD62G () as a function of PEG400. (C) Transitions with transient dwells at 15% PEG400 () and 30% PEG400 () have the same transition time, but different transient dwell durations. Grey lines indicating the power stroke velocity in the presence of 15% PEG400 have the same slope. Download figure D
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