Thermodynamic Characterization of RsiV Binding

ITC analysis was performed as previously described [12 (link)]. Briefly, 6xHis-2xFlag-RsiV^59–285 and mutants were purified as described above and buffer matched with HEW lysozyme (≥98% pure, Sigma Aldrich) by dialysis into 50 mM Na₂HPO₄, 200 mM NaCl, and pH 7.0 for 24 h at 4°C. Final protein concentrations as determined by absorbance at OD₂₈₀ were adjusted to 6xHis-2xFlag-RsiV^59–285 (0.01 mM) and HEW lysozyme (0.1 mM) with filtered dialysate. The protein samples were degassed and ITC measurements recorded using a MicroCal VP-ITC System (GE Healthcare) with HEW lysozyme as the injected sample and 6_XHis-2_XFlag-RsiV^59–285 as the cell sample. 21 injections of HEW lysozyme were used, with 180 seconds spacing between events. The chamber was kept under constant stirring at 350 rpm and all experiments were performed at 25°C. The binding reaction reached saturation during the experiment and control experiments where HEW lysozyme was injected into buffer showed that the heats of dilution were constant across all injections. The constant heat of dilution, as determined by the average of the last 3–5 injections, was subtracted and the data are analyzed using the single site binding model provided in the ITC analysis package. The values for affinity were averaged from three separate runs from two different protein preps.