Supplementary Section to “Motor step size and ATP coupling efficiency of the dsDNA translocase EcoR124I”

The phosphate release assay relies on a phosphate binding protein (PBP) covalently linked to a coumarin derivative (MDCC) that exhibits fluorescence that is sensitive to the specific, reversible association of Pi (Webb 2003; 2007). The use of MDCC-PBP as a phosphate sensor has been extensively characterised (Brune et al., 2001) and used widely in the study of motor proteins, including helicases (e.g., Dillingham et al., 2000; Martinez-Senac and Webb, 2005; Tomko et al., 2007). One of the advantages of the MDCC-PBP sensor is that Pi binding is rapid and tight: at 5 μM MDCC-PBP (pH 7.0, 5 °C), the observed rate of fluorescence change has a limiting rate of 150 /s and a KD of 0.1 μM (Webb 2003). Because we measured a steady-state accumulation of Pi, the observed rates were not limited by the KD and the signal was linear with Pi concentration at all temperatures (see below). The maximal ATPase rates measured under our experimental conditions (≤1200 μM ATP) were not rate-limited by the Pibinding rate (Webb, 2003). The purified and labelled sensor was characterised initially as described by Webb (2003) (Fig. S1). We chose to use 12 μM PBP in subsequent assays to allow reliable linear measurement of up to 6 μM Pi (all steady-state fits were made in the range of ≤5 μM Pi released). Calibration of the stopped flow was carried out for each temperature by titrating known concentrations of a Pi standard (Webb, 2003; Fig. S2; Table S1). In brief, Syringes “C” and “D” were filled with 5 μM MDCC-PBP in Reaction Buffer (50 mM Tris-Cl, pH 8.0, 10 mM MgCl2, 1 mM DTT). Equal volumes (50 μl) of C and D were mixed and the solution monitored for 60 s (Fig. S2A). 0.5 μM aliquots of the Pi standard (VWR International Ltd, Lutterworth, UK) were added to each syringe and the fluorescence re-measured each time after pushing the new solution into the observation chamber. The PMT response at each concentration of Pi