Figure 2.

PAN does not stall when it encounters the unfolded domain of GFP. (A,B) To determine mechanochemical coupling efficiency at ~Km levels of GFPssrA, ATP hydrolysis and GFPssrA unfolding (2 μM) were assessed concurrently, in the same well, using an NADH-coupled ATPase assay combined with GFPssrA unfolding (see Materials and Methods Section). Rate of ATP hydrolysis was measured by loss of NADH absorbance at 340 nm (A), while at the same time GFPssrA unfolding rate was measured by loss of fluorescence at ex/em: 485/510 (B). (C) Efficiency of mechanochemical coupling of ATP hydrolysis to GFPssrA was determined by plotting relative percentage ATPase and unfoldase onto a 2 dimensional plot and fitting with a line (R² = 0.9918). The dotted gray line is a hypothetical example of an ATPase that stalls (e.g., ClpX), where stalling is defined as <5% of the maximal degradation rate when the ATPase rate is 50% of maximal (Nager et al., 2011). (D–F) Same as (A–C), but at saturating GFPssrA substrate concentration (2 μM). Error bars are standard deviations from three independent experiments (n = 3).