Figure 3.

Determination of the affinity of BPI to bLPs. MST binding assay (A–D). Changes in movement in MST were monitored using BPI_N(A) NT647 incubated with increasing concentrations of the ligands [LPS EC (A), Pam₃CSK₄ (C)] or Pam₃CSK₄ Fluorescein incubated with increasing concentrations of BPI_N(A) (D). The binding affinity and r² values are indicated. The same assay using NT647-labeled rBPI was used to calculate K_D values for the interactions of the protein to LPS EC, LPS EC BL21, and (R)-Pam₃CSK₄ (B). NanoDSF was performed for rBPI incubated with the indicated ligands (E,F). Temperature-dependent change in fluorescence is indicated for the wavelength of 350 nm [E, upper part: absolute values, lower part: first derivate (f')]. Vertical lines indicate T_m. The shift in melting temperature (T_shift) caused by the ligands is shown (F). Temperature shifts above 1°C are interpreted as the influence of an interaction on the thermal stability of the protein. Data represent the mean of two (A,C,D) or three (B) technical replicates or means ± SD of two biological replicates (E,F).