Fig. 8.

In vitro choline-binding properties of point-mutated ChoX proteins. (A) [¹⁴C]choline binding was assayed with 5 μM ChoX and 5 μM [¹⁴C]choline. (B) Choline affinity constants (K_Ds) of ChoX variants. To calculate affinity constants, 5 μM ChoX protein was titrated with 0 to 50 μM [¹⁴C]choline, and bound [¹⁴C]choline (in disintegrations per minute) was plotted against the [¹⁴C]choline concentration (data not shown). The Michaelis-Menten equation was used for calculation of ligand-binding affinities. The data were fitted by nonlinear regression using SigmaPlot, version 9.0. Values are the means of three independent experiments ± standard deviations. (C) Circular dichroism analysis of the wild type and inactive ChoX variants. The spectrum of each protein with a concentration of 10 μM is plotted as ellipticity in millidegrees (mdeg) versus wavelength.