FIGURE 9.

Metal ion binding to SUMO-BDP determined by isothermal titration calorimetry. SUMO-BDP was expressed in a growth medium supplemented with 1 mm MnCl₂. The purified enzyme was exhaustively dialyzed against 50 mm HEPES, pH 7.5, 100 mm KCl, 5% glycerol, 5 mm EDTA, and 5 mm EGTA. The residual EDTA and EGTA were removed by a second dialysis step in the absence of the metal chelators. Individual or competitive titrations of SUMO-BDP with metal ions were performed as described under “Experimental Procedures.” The isotherms for three of the six globally analyzed ITC experiments are shown. They are Mg²⁺-only (black circles), Mn²⁺-only (gray squares), and the competition experiment (Mn²⁺ titrated into a 10:1 mixture of Mg²⁺ and SUMO-BDP, respectively; light gray diamonds). The results of the global fitting session are shown as the respectively colored lines.