Figure 6
Analysis of PLP binding equilibrium of wild type PNPO. (a) The figure shows the PLP binding curve obtained with 100 nM PNPO (protein subunit concentration). Reported data are the average ± standard deviation of five independent measurements. Equation (4) (see Methods) was fitted to these data. (b) Binding stoichiometry analysis obtained with 1 μM (black symbols), 2 μM (red symbols) and 4 μM (blue symbols) protein subunit concentrations. Fluorescence change, expressed as fractional variation as a function of the [PLPtot]/[PNPO] ratio, is linear as shown by the continuous line, up to the stoichiometry point corresponding to the crossing with the horizontal dotted line. The vertical dotted line through the stoichiometry point indicates that about one PLP molecules binds per enzyme subunit. (c) DSF measurements with wild type PNPO in the presence of different PLP concentrations. Fluorescence change is expressed as fractional variation as a function of temperature. The experiment was carried out using 2 µM enzyme and increasing PLP concentrations (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µM, as indicated by the arrow). The Boltzmann equation was fitted to thermal denaturation data to obtain melting temperatures. Each curve is the average of three independent experiments but, to clearly show the data, the standard error bars were not reported in the graph. In the upper inset, the saturation curve obtained by plotting the melting temperatures as a function of the PLP concentrations is shown. Data were analysed using the quadratic Eq. (5) (see Methods) to estimate the dissociation constant reported in Table 1. In the lower inset, the graph shows the first derivative (–dRFU/dT) of the fluorescence emission data reported in panel C. Images were generated using the software Prism 8 (GraphPad; https://www.graphpad.com/scientific-software/prism/).
