FIGURE 6.
ADP and AMPPNP increased TraI affinity for ssDNA. Titration of 50 nm FL-T17 with TraI was performed in the absence or presence of 350 nm T25 in buffer B (A), supplemented with 1 mm ADP (B) or 1 mm AMPPNP (C) at 25 °C. The solid lines are used to separate the data and do not have theoretical basis. D, model-independent binding isotherms of TraI to T25 in the absence or presence of 1 mm ADP or AMPPNP. The binding density and the concentration of free TraI were calculated from the titration curves in A–C using the MCT analysis (27). The solid lines are computer fits of the data using the Epstein combinatorial approach (Equation 9) under the condition m = 25. The intrinsic binding constants (Kint) for T25 in the absence of any nucleotide or in the presence of 1 mm ADP or AMPPNP are 2.8 (±0.4) ×107, 1.2 (±0.4) ×108, and 1.4 (±0.3) ×108 m−1, respectively.