FIGURE 4.

ApoCcmE differently recognizes class I and class II c-type apocytochromes. A, co-purification of His₁₀-apoCcmE with stoichiometric amounts of different c-type apocytochromes. The amounts of apoCcmE co-purified with apocytochrome c₂ (lane 1) was taken as 100% and compared with those seen with apocytochromes c′ and c₁ (lanes 2 and 3, respectively). B, co-purification of His₁₀-apoCcmE with apocytochrome c₁t39 and with its Cys-less derivative, apocytochrome c₁t39Cys/Ser*. The amounts of ApoCcmE co-purified with apocytochrome c₁t39 (lane 1) and its Cys-less derivative (Cys/Ser*) were determined as described in the legend for Fig. 3. C, real-time protein-protein interactions between Strep-Bt-apocytochrome c₂ immobilized on a SA-biosensor (ligand) and His₁₀-apoCcmE (analyte). The aligned sensorgram traces showing baseline (B) followed by association (A) and dissociation (D) steps were obtained using 400 nm Bt-apocytochrome c₂ and varying concentrations of apoCcmE. The raw data were fitted with high accuracy to a homogeneous 1:1 bimolecular interaction model, and the kinetic parameters were determined (Table 3).