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. 2018 May 25;5:47. doi: 10.3389/fmolb.2018.00047

Figure 3.

Figure 3

ITC of substrate and cofactor binding to TS. (A) Global fit of dUMP binding isotherms using two different syringe concentrations (open and filled circles) at 25°C. The modified general model (see text) was used for fitting (Sapienza et al., 2015). (B) Fits of dUMP binding isotherm at 5°C using two different models, the single site model with stoichiometry of two (open circles) and the modified general model (filled circles, which are shifted relative to open circles since the modified general model has cell concentration as a fitted parameter). Inset shows residuals for the two fits with modified general being clearly superior (Sapienza et al., 2015). (C) Thermodynamic parameters as a function of temperature for dUMP binding to free (filled symbols) and singly bound (empty symbols) TS. Note small but significant difference in slope of ΔH vs. T plots for the two binding events. (D) Raltritrexed binding to preformed C146S-dUMP complex at 15°C (Sapienza and Lee, 2016). Note the slope in the early part of the isotherm showing admixture of two processes with different ΔH values. Inset shows ΔH vs. T plot for first (filled circles) and second (empty circles) Raltitrexed binding events at three temperatures. Plots are parallel within error and show no signs of curvature in the narrow temperature range of measurements.