Figure 5.

Sequestration of unfolded GFP by ClpA. (A) GFP was acid-denatured as described in the text, and 40 pmol (50 μl) was added to reaction mixtures (100 μl final volume) containing buffer A with 20 mM creatine phosphate and 6 μg of creatine kinase in the absence of ClpA (green circles), in the presence of 160 pmol of ClpA and no nucleotide (black squares), in the presence of 160 pmol of ClpA and 10 mM ATP (red triangles), and in the presence of 160 pmol of ClpA and 1 mM ATP[γS] (open blue circles). Fluorescence was measured with time at 24°C. Fluorescence intensity of native GFP of the same concentration was taken as 1. (B) Reaction mixtures were as in A. Acid-denatured GFP was incubated for 5 min with 160 pmol of ClpA and 1 mM ATP[γS] and then 10 mM ADP (open black triangles), or 10 mM ATP (red squares) was added and incubations were continued. In a control, acid-denatured GFP was added alone to a reaction mixture (green circles).