Figure 6

BIAcore analysis of A1 binding to IsdB proteins. 6xHis-tagged IsdB NEAT1-NEAT2 or isolated NEAT1 and NEAT2 were immobilized on a NTA-Ni²⁺ sensor chip, while increasing concentrations of recombinant A1 (0–1.25 μM), lacking the 6xHis tag, were loaded in the mobile phase, following the single-cycle operation mode. Sensorgrams were subtracted from reference flow cell data and then fitted with the equation describing the heterogeneous ligand binding model to obtain the values of K_D and RUmax for the two ligand populations considered (L1 and L2) along with their relative abundance. IsdB NEAT1–NEAT2: K_D1 = 1.7 ± 0.3 μM; K_D2 = 0.9 ± 0.2 μM; RU_max1 = 354 ± 21; RU_max2 = 355 ± 23; L1 and L2 = 50%. NEAT1: K_D1 = 53 ± 2 μM; K_D2 = 5.3 ± 0.3 μM; RU_max1 = 948 ± 40; RU_max2 = 2705 ± 125; L1 = 26%, L2 = 74%. NEAT2: K_D1 = 0.3 ± 0.1 μM; K_D2 = 0.4 ± 0.1 μM; RU_max1 = 529 ± 51; RU_max2 = 527 ± 39; L1 and L2 = 50%. All measurements were carried out at 25 °C, in PBS containing 0.005% v/v Tween 20.