Itc200 microcalorimeter

ITC experiments were carried out using an iTC200 microcalorimeter (MicroCal). hcSHMT was equilibrated with binding buffer (50 mM HEPES pH 7.2, 100 μM EDTA), following PD10 gel filtration (GE Healthcare). Ligand stock solution (100 mM) was prepared by dissolving it in 100% DMSO. Titrations were carried out in 92.4% binding buffer, 10 mM glycine, and 1% DMSO. Aliquots (1.5 μL) of 0.5 mM or 0.3 mM LTX solution were injected into a solution of hcSHMT (37 μM) at 25°C. Binding of leucovorin to hcSHMT was assayed by titrating 27 μM hcSHMT with 1.5-μL aliquots of 1 mM leucovorin solution in 94% binding buffer, 10 mM glycine, at 25°C. The same titration was also carried out in the presence of 200 μM LTX in both protein and leucovorin solutions (in 92.4% binding buffer, 10 mM glycine, 1% DMSO). Data were corrected for heat changes from the injection of the ligand into the titration buffer and fitted using the “one-binding-site model” of the MicroCal version of ORIGIN. The heat of binding (ΔH), stoichiometry (n), and the dissociation constant (K_d) were then calculated from plots of the heat evolved per mole of ligand injected versus the molar ratio of ligand to protein using the software Origin provided by the vendor (MicroCal).

Acyl-CoA variants (C2–C12, all from Sigma-Aldrich) were used for isothermal titration calorimetry (ITC) experiments. ITC measurements were performed at 25°C using an iTC200 microcalorimeter (MicroCal). Proteins were dialyzed against a buffer containing 100 mM HEPES (pH 7.5) and 150 mM NaCl. All of the acyl-CoA variants (C2–C12) were dissolved in the same buffer. In each experiment, protein was titrated by either 16 injections of 2.45 μL of ligand solution or 26 injections of 1.5 μL of ligand solution, with 180 s intervals between two titrations. The experiment was performed in high-gain mode with the syringe rotating at 700 rpm. Data processing for ITC experiments was performed using the ORIGIN module of the iTC200 software.