1. Volume 5, Issue 4, Pages 639-648 (April 2000)
Dynamics of Substrate Denaturation and Translocation by the ClpXP Degradation Machine 
Yong-In Kim, Randall E. Burton, Briana M. Burton, Robert T. Sauer, Tania A. Baker 
Molecular Cell 
Volume 5, Issue 4, Pages (April 2000)
DOI: /S (00)

2. Figure 1
ClpX and ClpXPDFP Catalyze Denaturation but Not Degradation of GFP-SsrA
(A) Release of acid-soluble [35S]peptides from 0.25 μM [35S]GFP-ssrA following incubation with ClpXP or ClpXPDFP.
(B) Change in fluorescence at 511 nm of 0.21 μM GFP-ssrA following incubation with ClpXP or ClpXPDFP.
(A and B) The ClpX6 concentration was 0.3 μM, the concentration of modified or unmodified ClpP14 was 1 μM, and the concentration of GroEL14 trap was 10 μM. Gel filtration experiments confirmed that denaturated GFP-ssrA was bound to the GroEL trap when the latter molecule was present (data not shown).
(C) Rate constants for GuHCl denaturation of GFP-ssrA. The dashed line shows a semilogarithmic fit of the data r = 0.998; intercept = 6.4 × 10−8 s−1.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 Denatured GFP-SsrA Copurifies with DFP-Modified ClpP
(A) [35S]GFP-ssrA was denatured by ClpXPDFP and chromatographed on a Superdex 200 column. The elution positions of molecular weight standards are shown.
(B) SDS-PAGE of fractions 10–19.
(C) Autoradiograph of the gel shown in (B).
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3
Trapped GFP-SsrA Is Released from ClpXPDFP by Addition of a Second Substrate
(A) Time course of GFP-ssrA denaturation and second substrate–mediated renaturation. Denaturation reactions (100 μl) containing 0.2 μM GFP-ssrA, 0.3 μM ClpX6, and 1.3 μM ClpPDFP14 were allowed to proceed for ∼30 min at 30°C, and 20 μl of a second substrate (λcI-N-ssrA or λcI-N-ssrA-DD) was added to a concentration of 83 μM.
(B) Gel filtration (Superdex 200) of a sample 60 min after addition of λcI-N-ssrA, showing that trapped GFP-ssrA is largely released from ClpXPDFP. Reactions as in (A), except [35S]GFP-ssrA was used.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4
ATP and Second Substrate Directly Stimulate Release of Trapped GFP-SsrA
(A) Denaturation of GFP-ssrA by ClpXPDFP was carried out for 30 min (see Figure 3A legend), and reactions were diluted 10-fold into PD buffer (which contains ATP) with or without 0.3 μM ClpX and/or 25 μM λcI-N-ssrA. The fluorescence scale is 10-fold smaller for the data from 30–90 min to account for the dilution. In reconstruction experiments, the excess free ClpX present in this experiment had a negligible effect on the amount of native GFP-ssrA detected.
(B) Release of trapped GFP-ssrA from purified ClpPDFP complexes. Reactions contained 5 mM ATP or ATPγS, 2 μg of purified ClpPDFP·GFP-ssrA, 0.3 μM ClpX, with or without 25 μM λ cI-N-ssrA.
(C) Proposed pathway of substrate trapping by and release from ClpXPDFP. GFP-ssrA is denatured by ClpX and translocated to ClpP, where it resides stably enough to copurify by gel filtration. In the presence of ATP and additional substrate, ClpX actively promotes release of the denatured GFP-ssrA from ClpP into solution, where it can refold.
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5 Substrate Denaturation and Degradation Occur at the Same Rate
(A) Rates of degradation of [35S]GFP-ssrA by ClpXP were assayed at different substrate concentrations by the release of acid-soluble peptides (closed triangle), and rates of denaturation/degradation of unlabeled GFP-ssrA were assayed by changes in fluorescence (closed circle). The solid line is a fit of the combined data to a Michaelis-Menten model (r = 0.99; Km = 1.95 ± 0.26 μM; Vmax/ClpX6-total = 0.94 ± 0.04 min−1). (Inset) Time courses of the fluorescence and peptide release assays at ∼6 μM GFP-ssrA. Fluorescence change is shown by the small symbols and peptide release by the larger filled symbols. Predicted values for fluorescence and degradation, calculated from the kinetic model with denaturation as the slow step, are shown by the gray and black lines, respectively.
(B) Kinetic scheme for GFP-ssrA degradation. ATP is always present, but the ATPase cycle is not represented; for simplicity, the equilibrium for ClpX·ClpP complex formation is also not shown. Degradation is initiated by binding of the ssrA peptide tag of the substrate to the ClpX component of the ClpXP complex. In the slowest step of the overall reaction, ClpX catalyzes denaturation of bound substrate. Denatured protein is translocated to the ClpP chamber in a reaction that may be facilitated by ClpX-mediated opening of the axial pore of ClpP. The denatured and translocated protein is degraded to peptide fragments by the ClpP active sites. Following degradation, peptides are released from the complex. Although not shown, based on its stimulation of protein release from ClpPDFP, ClpX is likely to also facilitate this release reaction. The assay used to measure degradation will detect both free peptides and those still bound to ClpP; therefore, we have not measured release, independent from degradation.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6
Rate of Release of Trapped GFP-SsrA Depends on the Second Substrate Concentration and the Degradation Signal
Denaturation reactions (100 μl) containing 0.3 μM ClpX6, 0.8 μM ClpPDFP14, and 0.2 μM GFP-ssrA were performed for 30 min at 30°C; different concentrations of λcI-N-ssrA (closed triangle), Arc-ssrA (closed circle), or Arc-MuA10 (closed square) were added (in 20 μl); and initial rates of GFP-ssrA release/refolding (see Figure 3A and Figure 4A) were determined by linear regression of fluorescence data. The rates shown were obtained by dividing the initial rates by the concentration of ClpX6PDFP14·GFP-ssrA (13.6 pmol/120 μl), as estimated from the fluorescence value at the time of addition of the second substrate. The solid lines are fits to the equation: rate = Vmax·(1 + K1/2/[second substrate])−1.
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7 Inhibition of GFP-SsrA Denaturation by SsrA Peptides
Kinetics of ClpXPDFP denaturation of GFP-ssrA in the presence of no peptide (−), wild-type ssrA peptide (ssrA), carboxamide ssrA peptide (CONH2), and the AA→DD ssrA peptide derivative (see text). Reaction mixtures with 0.3 μM ClpX6 and 0.8 μM ClpPDFP14 were preincubated for 2 min at 30°C, and a mixture of GFP-ssrA (1.3 μM) and peptide (280 μM) was added. Inset shows a Lineweaver-Burk plot for the inhibition of GFP-ssrA denaturation by the wild-type ssrA peptide and λcI-N-ssrA, both at 13 μM.
Molecular Cell 2000 5, DOI: ( /S (00) )