Itc200

The binding affinity of Spy H96L for Im7 6–26 peptides was determined by isothermal titration calorimetry (ITC) using a MicroCal iTC200 instrument (Malvern). The thermodynamic parameters upon the titration of Spy H96L (1.25 mM) with the Im7 6–26 peptides (0.11 µM for the wild type, 0.055 µM for the Im7 6–26 peptides L18pI-Phe, L19pI-Phe and K20pI-Phe) were measured at 10°C in a buffer consisting of 40 mM HEPES–NaOH pH 7.5, 100 mM NaCl. For each experiment, an injection volume of 2 µl was used at intervals of 180 s. The ITC thermograms were fitted to a one-site model using the Origin software provided with the instrument. The Im7 6–26 peptide binds to Spy H96L with a dissociation constant of 7.8 ± 0.6 µM, which is similar to the dissociation constant of a partially folded variant of Im7 and wild-type Spy. The Im7 6–26 peptides L18pI-Phe, L19pI-Phe and K20pI-Phe bind to Spy H96L with dissociation constants in the same range as the wild-type Im7 6–26 peptide (Supplementary Fig. S1).

Purified PXR(130-434)-SRC-1 was dialysed overnight against Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM using 10 kDa molecular weight cut-off dialysis cassettes (Slide-A-Lyzer 0.5 ml 10 K MWCO, Thermo Scientific). Protein concentration was determined spectrophotometrically (ɛ_280 nm=26,210 l mol⁻¹ cm⁻¹). Duplicate experiments were performed on Microcal ITC200 (Malvern) operating at 25 °C. Titrations were carried out in Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM supplemented with 0.05% Tween 20 and 5% DMSO (syringe, sample and reference cells). PXR (5 μM) was disposed in 200 μl cell and compounds were delivered from 40 μl syringe. Compound solutions were set to 300 μM when tested individually (Fig. 5b,c), 50 μM each when used simultaneously (Fig. 5f) and 50 μM (EE2, Fig. 5d) or 200 μM (TNC, Fig. 5e) when tested after pre-incubation of PXR with 50 μM TNC or EE2, respectively. Heat exchanges were monitored throughout titrations consisting of 19 injections (one time 0.5 μl followed by 18 times 2 μl) of compound solutions into the cell containing PXR solution. Data analysis and thermodynamic parameter fitting used Microcal Origin software (Malvern).

ITC experiments were performed on an iTC200 (Malvern Instruments) at 10 °C in 50 mM potassium phosphate buffer, pH 7.5. The PGKETQL peptide was titrated into a solution of pWT PDZ2 L391F. The experiments were designed such that the C values were within 1–1000 (C value = N × [Protein]/K_d, where N is the stoichiometry of the interaction, [Protein] is the molar concentration of protein in the cell and K_d is the equilibrium dissociation constant). The software provided by the manufacturer was used to determine the thermodynamic parameters of the peptide/PDZ interactions using nonlinear least square fitting assuming a 1:1 model. Since a clear saturation was reached in the experiments we corrected for the small heat of dilution by subtracting integrated peaks until a minimum in χ2 was obtained.

The protein was buffer exchanged into 50 mM HEPES, 100 mM NaCl, 2 mM MgCl₂, and 1 mM TCEP at pH 7.4. The protein was concentrated to 50–100 µM, and DMSO was added to a final concentration of 5% (^V/_V). The fragments were dissolved in DMSO to yield a 100 mM stock solution, which was further diluted with buffer to yield a 5 mM solution also containing 5% (^V/_V) DMSO. A MicroCal iTC200 (Malvern) instrument was used with the measuring cell set to 25°C, while the cooling jacket was set to 15°C. After a 120 s initial delay following temperature equilibration, a first injection with 0.5 µl over 2 s was done. Nineteen injections with 2 µl over 4 s were performed every 180 s (Ruhmann et al., 2015 (link)). The measurement was conducted using a needle stirring speed of 1,000 rpm and a reference heat rate of 10 μcal/s. Experiments were aborted, when the measuring cell was unable to reach a heat rate greater than 9 μcal/s during equilibration. Affinities were averaged from duplicates.

The interactions of the NRP1-b1 domain with ClTx and EG00229 were performed using a Microcal ITC200 (Malvern) in buffer containing 50 ​mM sodium citrate pH 5.5, 150 ​mM NaCl at 32 ​°C. To test the binding, 800 ​μM of ClTx or EG00229 were titrated into a sample cell containing 40 ​μM of NRP1-b1 domain. Competition assays were carried out by injecting 800 ​μM of ClTx into 40 ​μM of NRP1-b1 domain +200 ​μM of EG00229. Each experiment involved an initial 0.4 ​μL (excluded from data processing) followed by series of 12 injections of 3.22 ​μL each with a stirring speed of 750 ​rpm and a spacing period of 180 ​s between injections. In all binding experiments the equilibrium dissociation constant (K_d), change in binding enthalpy (ΔH), change in Gibbs free energy (ΔG) and entropy change (ΔS) were analysed after fitting data to a single-site binding model with stoichiometry (N) set to 1.0 for calculation.