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. 2012 May 16;287(28):23819–23829. doi: 10.1074/jbc.M112.371492

FIGURE 1.

FIGURE 1.

Binding of IIAChb*(H89E) to 15N-labeled HPr. Backbone amide chemical shift perturbations upon titrating unlabeled IIAChb*(H89E) into a solution of 15N-labeled HPr at 20 °C. The chemical shifts were monitored using 1H-15N HSQC spectroscopy at a spectrometer 1H frequency of 600 MHz. ΔδH/N = [(Δδ15N)2/25 + (Δδ1H)2)/2]1/2 in parts per billion (ppb) (69). The IIAChb*(H89E):HPr molar ratios, expressed in terms of subunit concentration of IIAChb*(H89E), are 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0, with corresponding concentrations of 0.0 0.24, 0.47, 0.68, 0.87, 1.05, and 1.23 mm in subunits for IIAChb*(H89E), and 0.50, 0.48, 0.47, 0.45, 0.44, 0.42, and 0.41 mm for HPr. The solid lines represent the results of a global nonlinear least squares best fit to all the titration data simultaneously, using a simple equilibrium binding model. The optimized KD value is 0.7 ± 0.1 mm.