FIGURE 2.
Production of degradation intermediate increases with increasing length of a GAr. A, substrates (Cy3-labeled) of the form GST-I27-GArN-GFP-ssrA, containing GAr sequences with n = 7, 8, 9, 10, or 15 amino acids were degraded by ClpXP for 60 min, and samples were periodically analyzed. Samples were separated by SDS-PAGE and scanned by fluorescence imaging, using fluor-specific excitation and emission wavelengths. Cy5-labeled BSA included in each sample was fractionated on the same gel and analyzed as a loading control (lower portion of each panel). The position of full-length substrate (solid arrows) or degradation intermediate (dashed arrows) is indicated. B, the abundance of the full-length substrate (Parent) and degradation intermediate in A were quantified, normalized to the intensity of the Cy5-BSA bands in the same lane. The values for substrate with GAr of indicated length and the corresponding degradation intermediates are expressed as a percentage of the total of the full-length parent protein present at t = 0. C, the molar yield of intermediate as a percentage of the decrease in parent substrate was calculated from the mean of 15, 30, and 60 min time points, as described under “Experimental Procedures.” Means ± S.D. (error bars) of three independent experiments are plotted.