For NMR, all samples were dialyzed in NMR buffer 50 mM potassium phosphate (pH 6.8), 140 mM NaCl and 1 mM β-mercaptoethanol). NMR experiments were performed on Varian 800- and 600-MHz and Bruker 700- and 600-MHz spectrometers. Abl kinase domain (Abl KD) was expressed and purified as described before (41 (link), 42 (link)). Purified and Tev-processed Crk II was phosphorylated by the addition of catalytic amounts of Abl KD in buffer (50mM KPi (pH 6.8), 150mM NaCl, 1mM β-mercaptoethanol) supplemented with 5 mM ATP and 10 mM MgCl2. The reaction was carried out at room temperature. Stoichiometry of phosphorylation at Y221 was >95% by NMR.
All calorimetric titrations were performed on an iTC200 microcalorimeter (GE). Unphosphorylated or phosphorylated CrkII was at 40 µM in the sample cell and the PPII ligand was at 400 µM in the injection syringe. Ligand was dissolved in the flow through of the last buffer exchange. Data for the preliminary injection, which are affected by diffusion of the solution from and into the injection syringe during the initial equilibration period, were discarded. The data were fitted with Origin 7.0.
All calorimetric titrations were performed on an iTC200 microcalorimeter (GE). Unphosphorylated or phosphorylated CrkII was at 40 µM in the sample cell and the PPII ligand was at 400 µM in the injection syringe. Ligand was dissolved in the flow through of the last buffer exchange. Data for the preliminary injection, which are affected by diffusion of the solution from and into the injection syringe during the initial equilibration period, were discarded. The data were fitted with Origin 7.0.