1. Volume 155, Issue 3, Pages 636-646 (October 2013)
The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears 
Maya Sen, Rodrigo A. Maillard, Kristofor Nyquist, Piere Rodriguez-Aliaga, Steve Pressé, Andreas Martin, Carlos Bustamante 
Cell 
Volume 155, Issue 3, Pages (October 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Single-Molecule Experimental Constructs
(A) Cartoon depicting protein unfolding and polypeptide translocation by ClpXP.
(B) Experimental geometry of dual-trap optical tweezers assay (not to scale). Biotinylated ClpX was immobilized on streptavidin-coated beads (SA). The DNA-tethered substrate was immobilized on the surface of beads coated with antidigoxigenin antibodies (AD).
(C and D) Single-molecule trajectories of substrate processing by ClpXP at 1 mM ATP and forces ranging from 6 to 12 pN. GFP unfolding events are indicated by arrows and followed by translocation of unfolded polypeptide. Substrates are composed of GFP moieties (green) fused to titinCM and a C-terminal ssrA tag (black and red, respectively), as well as an N-terminal ybbR tag (light blue) for attachment to the bead. Raw data (2.5 kHz in gray) were filtered and decimated to 100 Hz (green, black, and blue lines).
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4. Figure 2 Effects of ATP, ADP, and Pi on Translocation
(A) General scheme depicting a motor (M) that binds to one ATP molecule (T), undergoes a tight binding transition, and hydrolyzes ATP, followed by the release of inorganic phosphate (Pi) and ADP.
(B) Representative trajectories for translocation of the titinCM moiety of the fusion substrates measured between 6 and 12 pN at different ATP concentrations with ATP regeneration system (ATP/RS). The trajectories are offset for clarity.
(C) Pause-free velocity of translocation (mean ± SEM) as a function of external force at 5 mM ATP (red symbols) and 35 μM ATP (black symbols).
(D) Km (blue) and Vmax (green) are plotted against force. Inset: Km/Vmax ratio plotted for forces between 5 to 15 pN. Error bars are from the fits (SEM).
(E) Km (blue) and Vmax (green) plotted against ADP concentration at 7.5 pN. Error bars are from the fits (SEM).
(F) Pause-free velocity of translocation (mean ± SEM) plotted as a function of phosphate concentration [Pi] at 7.5 pN with a fixed [ATP] (see also Figure S1).
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5. Figure 3 ATPγS Induces Long-Lived Pauses
(A) Representative trajectories for forces between 6 and 12 pN were measured with increasing [ATPγS] at fixed [ATP]. Trajectories are offset for visual clarity.
(B) Translocation rate (mean ± SEM) plotted against [ATPγS], with the fit shown in red.
(C) Inverse density of ATPγS-induced pauses (mean ± SEM) plotted against the inverse of [ATPγS], with the linear fit shown in red. Inset: pause density (mean ± SEM) plotted as a function of [ATPγS].
(D) Pause duration (mean ± SEM) as a function of [ATPγS] (see also Figure S2).
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6. Figure 4 Effect of ATP Concentration on Burst Size and Dwell Duration
(A) Representative trajectories of ClpXP translocating substrate in 3 nm steps at 10–14 pN and different ATP concentrations. Raw data were filtered and decimated to 1,250 (in gray) or 50 Hz (in red, blue, green). t test fits to the data are shown in black.
(B) Burst size distributions for ATP concentrations near Km (red) and saturating ATP (blue).
(C) Mean dwell duration (±SEM) plotted against [ATP]. Inset: dwell-time distribution for near-Km conditions (red) and saturating ATP (blue) (see also Figure S3).
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7. Figure 5 GFP Unfolding Mechanism
(A) Probability of GFP unfolding as a function of [ATP] and ATPase rate (inset). Error bars are ± SEM.
(B) GFP unfolding events display two intermediates at 300 Hz.
(C) Mechanism of GFP unfolding by ClpXP at [ATP] ≫ Km.
(D) Trajectory at [ATP] = 200 μM illustrating the ClpXP-induced unfolding and refolding of β11 (see also Figure S4).
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8. Figure 6 Minimal Mechanochemical Model of ClpXP Translocation
(A) The pathway of ATP hydrolysis for a single subunit of ClpX. An empty subunit (E, red sphere) binds ATP (T, orange) and undergoes a tight binding of ATP (T∗, green). Then, the subunit hydrolyzes ATP to ADP and Pi (DP, blue), the force-generation step occurs upon phosphate release (D, purple), and ADP dissociates, leaving an empty subunit (E, red sphere).
(B) Schematic depiction of intersubunit coordination at saturating (green box) and limiting ATP concentrations (red box) for one possible scenario depicting sequential ATP binding. The subunits in gray correspond to those that do not bind ATP. During the dwell phase, at least two ATPs are bound to the high-affinity subunits (T, blue outline), and additional ATPs can bind to the low-affinity ClpX subunits (T, green outline), depending on [ATP]. During the burst phase, the motor hydrolyzes all bound ATPs, releases phosphate, and translocates the substrate by 2, 3, or 4 nm into the central pore.
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9. Figure S1
Nucleotide Dependence of ClpX Translocation Velocity, Related to Figure 2
(A) Pause-free velocity is shown as a function of [ATP] at 7.5 pN resisting force. Each data point represents the average of the pause-free velocities from a group of individual trajectories (n = 8-50) with SEM error bars. Data were fit to a simple Michaelis-Menten equation with Km = 31 ± 6 μM (SEM) and Vmax = 8.7 ± 0.4 nm/s (SEM).
(B) Pause-free velocity (mean ± SEM) plotted against [ATP] at [ADP] = 10 μM (black) or [ADP] = 50 μM (purple), with [Pi] held constant at 5 μM. Each point is the mean velocity from a set of individual traces (n = 7-30) measured at 7.5 pN resisting force. The data fit well to a Michaelis-Menten equation (solid lines in black and purple).
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10. Figure S2
Rate of ClpXP ATPγS Hydrolysis Measured by Thin-Layer Chromatography, Related to Figure 3
(A) Thin-layer chromatography assay of 35S-labeled ATPγS hydrolysis by ClpXP. 35S-ATPγS was incubated with 0.3 μM ClpX and 1.5 μM ClpP (top) or buffer only (bottom) for the time indicated, before being quenched with 2.5 volumes of stop buffer and spotted on TLC plates (see Extended Experimental Procedures). The positions of ATPγS and PO3S are indicated.
(B) Rate of ClpXP-mediated hydrolysis of ATPγS as a function of [ATPγS] in the presence (red) and absence (blue) of titinCM-ssrA. Conditions were as in (A) with the addition of 10 μM titinCM-ssrA as indicated. Fitting the data to the Michaelis-Menten equation provided the values of kcat = 6.3 ± 0.9 min−1 ClpX−1 and KM = 29 ± 10 μM in the presence of titinCM-ssrA, and kcat = 2.5 ± 0.2 min−1 ClpX−1 and KM = 6.2 ± 2.0 μM in the absence of titinCM-ssrA. All fitted values are mean ± SEM.
(C) Rate of ATP hydrolysis by ClpXP is shown as a function of [ATP] in the presence (red) and absence (blue) of titinCM-ssrA. Fitting the data to the Michaelis-Menten equation provided the values of kcat = 497 ± 20 min−1 hexamer−1 and Km = 57.2 ± 6.1 μM in the presence of titinCM-ssrA, and kcat = 111 ± 13 min−1 hexamer−1 and Km = 58.9 ± 2.0 μM in the absence of titinCM-ssrA. All fitted values are mean ± SEM.
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11. Figure S3
Translocation Burst Size Is Dictated Mostly by the Geometry of ClpX and Not by Regularities of the Polypeptide Substrate, Related to Figure 4
(A and B) Distribution of burst sizes occurring between 6-10 pN (red line) and pN (blue line).
(A) Burst size is shown in units of extension (nm), reflecting the end-to-end distance of polypeptide translocated during a single burst phase. The distributions of burst sizes in nanometers within each force range were determined to be identical with p = 0.72 (two-sample Kolmogorov-Smirnov test, null hypothesis: both distributions identical).
(B) Burst size is shown in units of contour length in amino acids, reflecting the number of amino acids that pass through the ClpX pore during a single burst phase. The distribution of burst sizes in amino acids within each force range was determined to be different with p = 1.5e-6 (two-sample Kolmogorov-Smirnov test, null hypothesis: both distributions identical).
Cell  , DOI: ( /j.cell )
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12. Figure S4
Detection and Characterization of Intermediates during GFP Unfolding, Related to Figure 5
(A) The rips (sudden increases in bead extension) during GFP-unfolding revealed three well-defined transitions, indicating the presence of two unfolding intermediates, labeled I and II, at saturating ATP concentrations. The second intermediate was observed in 90% of all traces (top panels), while both intermediates were present in only 40% of them (top left panel). We rarely observed traces displaying only the first intermediate or no intermediates at all (∼10%, bottom panels).
(B) Distribution of lifetimes for the folded state, and first and second Intermediates during GFP unfolding by ClpXP. The leftmost panel shows the distribution of lifetimes of the folded state, which corresponds to the GFP unfolding time. The distribution of lifetimes for the first and second intermediate (middle and right panel, respectively) was well fitted by a single-exponential function. The first intermediate has a lifetime of 45 ± 10 ms, whereas the second intermediate exhibits a lifetime of 130 ± 15 ms. All fitted values are mean ± SEM.
(C) Plot of force versus change in extension for the transition to the first intermediate F → I (blue), the second intermediate I → II (red), the unfolded state II → U (green), and the sum of all transitions F → U (black). For clarity, the shift in orientation of the folded structure has been removed (see Extended Experimental Procedures, Equation S14).
(D) Mapping the measured changes in contour length for each intermediate transition to the extracted structural elements of GFP (see Extended Experimental Procedures). Top: root-mean-square-deviation (RMSD) of the measured extension changes from the predicted WLC extension change as a function of linker residue position for the transition to the first intermediate (left) and the second intermediate (right). Bottom: GFP structural topology showing the identified structural elements. The transition to the first intermediate is identified to be the extraction of β11, and the transition to the second intermediate corresponds to the extraction of β10-7.
(E–G) Histograms of contour-length change for the transitions: (E) from the folded state to the second intermediate F → II, (F) from the second intermediate to the unfolded state II → U, and (G) the sum of these transitions F → U.
(H and I) Lifetime distribution of the second intermediate when the preceding transition was observed to be (H) I → II, or (I) F → II.
(J and K) Distributions of the change in contour length and the mean time constant for unfolding and refolding events of β11. These events were observed in ∼20% of all traces at ATP concentrations near Km, and ∼11% of traces at intermediate [ATP] =  μM. At [ATP] > 200 μM, we hardly detected these events (less than ∼1% of all traces). (J) Distribution of changes in extension upon unfolding and refolding of β11. (K) β11 refolding time fits well to a single-exponential with a τ = 0.23 ± 0.08 (mean ± SEM).
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