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. 2001 Mar 20;98(7):3768–3772. doi: 10.1073/pnas.071043698

Figure 5.

Figure 5

FRET between donor-labeled EDANS-miClpP and acceptor-labeled Fl-LYssrA initiates earlier during a ClpA-mediated translocation reaction for a substrate probe that is adjacent to the COOH-terminal ssrA tag. ClpA (3 μM hexamer), miClpP (1.5 μM tetradecamer), either Fl-LYssrA (1.5 μM), and ATPγS (1 mM) were incubated together in reaction buffer at 25°C for 45 min, then rapidly mixed with an excess of ATP (10 mM) in the stopped-flow apparatus. Each trace is the sum of 10 runs. The difference in the final FRET efficiency reached with the two molecules (18% vs. 24.5%, respectively) likely results from asymmetric binding of the tagged substrate within the ClpP chamber and, hence, different distances and relative orientations between the probes.