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.