Single Turnover ClpB Catalyzed Protein unfolding. A) RepA(1-70)-Titin1 (Blue), RepA(1-70)-Titin2 (purple), RepA(1-70)-Titin3 (green). Each construct from N-to C-terminus consists of the first 70 amino acids of the Phage P1 RepA protein, a known binding sequence for ClpB followed by tandem repeats of the Titin I27 domain separated by linkers. Each construct contains a single cysteine shown in space-filling at the C-terminus that has been reacted with Alex Fluor (AF)-555. B) Schematic of steps in forming pre-bound complex based on our previous work (22-25). ClpB (black) is assembled into hexameric rings competent for substrate binding by adding ATPγS, illustrated as bound to the RepA-Titin1 substrate in blue, followed by rapid mixing with ATP. As shown, ClpB is expected to unfold the Titin I27 domains and translocate the newly unfolded substrates through the axial channel of the hexameric ring. C) Schematic representation of stopped-flow. Syringe 1 contains the indicated concentrations of ClpB monomer, ATPγS, and RepA-TitinX, where x = 1, 2, 3. Syringe 2 contains 400 μM ATP and 20 μM α-casein to serve as a trap for any free ClpB. The contents of the two syringes are rapidly mixed at a 1:1 mixing ratio and flow into the observation channel where AF555 is excited at λex = 555 nm and emission is observed at λem >570 nm. D) Representative time-course collected using strategy in (C) using RepA-Titin3 after pre-incubating the sample at 25 °C for 600 s. E) Successive experimental time-courses collected as in D. The total time of incubation before collection of the time-course is indicated.

Test for ATPγS driven Protein Unfolding by ClpB. A) Mixing strategy as in Fig. 1 C but with ATP replaced with 2 mM ATPγS in Syringe 2. B) Time-courses collected using RepA-Titin1(blue), RepA-Titin2(purple), and RepA-Titin3(green) plotted as relative fluorescence enhancement vs. time. The solid line represents the best-fit line from fitting to Scheme 1, Fig. 4 A. The fitting parameters obtained are the unfolding rate constant, kU = (0.0042 ± 0.0003) s-1, and the kinetic step-size, m = 26 (21, 28) aa. C) length of substrate vs. peak time determined from B. The plot was fit to a linear equation to yield a slope = (0.098 ± 0.003) aa s-1 and intercept of (71 ± 7) aa.

Sequential Mixing Stopped-Flow Strategy. A) Schematic representation of sequential mixing. Syringe 1 contains 8 μM ClpB monomer, Syringe 2 contains 2 mM ATPγS and 200 nM RepA-TitinX, Syringe 3 contains 40 μM α-casein and 1 mM ATP. The contents of Syringes 1 and 2 are mixed 1:1 in mixing chamber 1 leading to a concentration of 4 μM ClpB, 1 mM ATPγS, and 100 nM RepA-TitinX. The sample ages for a user-defined amount of time, Δt1, followed by rapid mixing with the contents of Syringe 3. The sample flows into the observation channel at a final concentration of 2 μM ClpB, 50 nM RepA-TitinX, 500 μM ATPγS, 500 μM ATP, and 20 μM α-casein. In the observation channel AF555 is excited at λex = 555 nm and emission is observed at λem >570 nm. B) Representative time-courses from the average of five or more sequentially collected time-courses for RepA-Titin1(blue), RepA-Titin2(purple), and RepA-Titin3(green) at Δt1 = 600 s. The solid lines represent the best-fit line from fitting to Scheme 1. The fitting parameters obtained are kU = (0.017 ± 0.002) s-1 and m = (56.5 ± 0.7) aa. C) Total length of substrate as a function of peak time determined for Δt1 = 300, 420, and 600 s. Solid lines represent weighted linear fits yielding D) slope vs. Δt1 and E) the intercept vs. Δt1. All data points and error bars represent the average and standard deviation determined from three replicates.

Parameters obtained from model-independent analysis on experiments presented in Fig. 3 and Supp. Fig. 5.

n-step sequential mechanism for 500:500 μM ATP:ATPγS. A) Proposed kinetic scheme for ClpB catalyzed protein unfolding and translocation on RepA-TitinX substrates. Parameters B) kU C) m D) mkU obtained from fitting to scheme 1 at each Δt1 are shown in solid black circles. The black solid line represents the best weighted fit line to a linear equation with zero slope to yield the average value of the unfolding rate constant, kU = (0.017 ± 0.002) s-1, kinetic step-size, m = (56.5 ± 0.7) aa, and overall rate, mkU = (0.89 ± 0.09) aa s-1.

Parameters obtained from global fitting the time-courses obtained from experiments in Fig. 3 and Supp. Fig. 5 to Scheme 1