Unfolding of GFP-SsrA by ClpX. (A) GFP-SsrA (5 μM) was incubated at 37°C with various amounts of ClpX plus 5 mM ATP and an ATP-regenerating system. Fluorescence traces with no ClpX (●) and 0.2 (⋄), 0.5 (□), 1.0 (○), and 3.5 μM ClpX (▵) are shown. (B) GFP-SsrA (1 μM) was incubated at 37°C with 1–2 μM ClpX and 5 mM ATP; a typical result of unfolding with 1 μM ClpX is shown (○). As a control, a competitive substrate, 7 μM λO, was mixed with 1 μM ClpX before adding the GFP-SsrA (□). At the arrow, one of the following was added to separate unfolding reaction mixtures: 8 μM λO (▴), 10 μM GroEL-trap (●), or 8 μM λO and 10 μM GroEL-trap (■).