Figure 3

Mutant rPhl p 7 lacks calcium-binding capacity, but represents a folded protein with slightly reduced thermal stability. Size exclusion chromatography coupled to ICPMS was performed with wildtype (A) and mutant (B) rPhl p 7. UV chromatograms (VWD signal) at 230 nm (grey lines) are given in milli arbitrary units (mAu, right y-axis) and the corresponding calcium ion count mass spectrometry traces (m/z 40, black lines, left y-axis) are shown in kilocounts per second (kCPS). The x-axis represents the elution time in minutes (min). (C) CD spectra of wildtype (WT, black) and mutant (Mut, grey) rPhl p 7 recorded under native conditions (continuous lines) or after addition of EDTA (dotted lines). Note that the rPhl p 7 Mut ETDA line is fully congruent with the rPhl p 7 Mut line. (D) Temperature ramping experiments from room temperature (continuous lines) to 95 °C (dotted lines) and back to room temperature (dashed lines). The spectra are expressed as the mean residue ellipticity (deg*cm²*dmol⁻¹; y-axis) at a given wavelength (x-axis).