FIG 4.
Binding of PilA2 to ComZ. (A) Size exclusion chromatography analysis. (Upper) Elution profiles (absorption at 280 nm) of ComZ-PilA2 mixture (profile A), ComZ alone (profile B), and PilA2 alone (profile C). Separation was carried out on a HiLoad 16/600 Superdex 200 PG column (GE Healthcare), with a flow rate of 1 ml/min in 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol. ComZ (0.036 μmol) and PilA2 (0.023 μmol) were loaded. (Lower) SDS-PAGE of eluted peaks from each SEC run. (B) Titration of PilA2 into ComZ using isothermal titration calorimetry (ITC). Two-μl aliquots of recombinant PilA2 (314 μM) were titrated into the sample cell containing 300 μl of ComZ (33 μM). (Lower) Raw data with fitted baseline. (Upper) Data fitted assuming a single binding site. Fitted parameters are the following: stoichiometry (n), 1.1; equilibrium binding constant (Kd), 0.69 μM; enthalpy change (ΔH), 2.3 kcal/mol; ΔG = −8.4 kcal/mol; TΔS = 10.7 kcal/mol.