Fig. 6.

(A) Michaelis-Menten plots of ClpXP degradation (0.1 μM ClpX₆; 0.2 μM ClpP₁₄) of ssrA-tagged variants of GFP and ^SFGFP in the presence of 4 mM ATP and an ATP-regeneration system. Initial degradation rates were calculated from changes in 467-nm fluorescence. The lines are fits to the equation rate = V_max•[S]/(K_M + [S]). Error bars (± 1 SD) based on four independent replicates. K_M and V_max values for each substrate are listed in Table 1. (B) Proteins (0.5 μM) were incubated with different concentrations of GuHCl for 2 weeks, and denaturation was assayed by 467-nm fluorescence. The solid lines are fits to a two-state unfolding model. cp7-^SFGFP-ssrA has an unusual native baseline and higher m_eq value than the other proteins (Table 1). These properties could reflect an increase in the solvent accessibility of the unfolded protein and/or the presence of a populated unfolding intermediate. (C) Rates constants for unfolding (k_u) were determined by single-exponential fits of changes in 467-nm fluorescence after jumps to different concentrations of GuHCl. The values plotted are averages of three independent experiments ± 1 standard deviation. The final protein concentration in each assay was 0.5 μM.