Itc200

Isothermal titration calorimetry (ITC) was conducted using an ITC200 calorimeter (Microcal). UbcH7(C86K)-Ub isopeptide conjugate (500 µM) was titrated into 50 µM RNF216 RBR-helix (511-784, C688A, pSer719) or HOIL-1 helix-RBR (233-510, C460A) with or without 150 µM di-Ub. The phosphorylated RNF216 construct was used here as this was previously shown to be most active in E2-Ub discharge assays^{23 (link)}. All titrations were performed with 1 × 1 µl and 19 × 2 µl injections at 25 °C, with both components in 10 mM HEPES pH 7.9, 100 mM NaCl. Data were collected using ITC200 software (GE Healthcare) and processed and analysed using Origin software (Microcal).

Measurements were performed using an ITC₂₀₀ (GE Healthcare) at 298 K. For the direct titrations of inhibitors 3 and 4, the cell was filled with a 100 µM solution containing 67% active ALR-2, an excess of NADP⁺ (0.25 mM), and 3% (v/v) DMSO in 10 mM HEPES buffer at pH 8.0. For the ITC measurements, the oxidized cofactor NADP⁺ was added, which also binds to the protein but can no longer be converted. This prevents falsification of the heat signal of the inhibitor binding by chemical reactions of the cofactor, as demonstrated in previous studies [15 (link)]. The syringe was filled with the same concentration of NADP⁺ and DMSO along with inhibitor (0.75–1.0 mM) in 10 mM HEPES at pH 8.0. An initial injection of 0.3 µL, which was excluded from the analysis, was followed by 27 inhibitor injections of 1.3 µL with a duration of 0.6 s and an interval of 180 s between each injection.
For the displacement titrations of the weak inhibitors 5 and 6, these were additionally added to the cell according to a saturation of the protein at approximately 94% and then titrated with the strong inhibitor 9. Here, one injection of 0.3 µL was followed by 37 injections of 1.0 µL with a duration of 0.6 s and an interval of 180 s between each injection.

The interaction of the ADAM10 peptide or a control peptide (KT peptide) with scrambled sequence with PC or PS liposomes was analysed by ITC measurements on an ITC200 (GE Healthcare) (Brandenburg et al., 2005 (link)). Briefly, 0.5 mM solutions of the ADAM10 peptide in 5 mM HEPES were titrated 20 times in 2 μl injections to 250 μl 0.1 mM PS or PC liposome dispersions — leading to a 4:3 lipid:protein molar ratio after the last injection. The heat of dilution was determined in control experiments by injecting peptide solution into buffer (5 mM HEPES, pH 7.4). Enthalpy changes were recorded over time; measurements were performed at 37°C.

All experiments were performed in a buffer containing 150 mM NaCl, 20 mM HEPES 7.4, and 2 mM BME. Peptides were synthesized and delivered as lyophilized powder (Peptide Specialty Labs GmbH) and dissolved directly in buffer. The peptides were centrifuged at 14,000 × g for 10 min, and only the supernatant was used. The dissolved peptide concentrations were calculated based upon their absorbance at 280 nm and their corresponding molar extinction coefficient. Typical experiments consisted of titrations of 20 injections of 2 μl of titrant (peptides) into the cell containing COP1 at a 10‐fold lower concentration. Typical concentrations for the titrant were between 500 and 3,000 μM for experiments depending on the affinity. Experiments were performed at 25°C and a stirring speed of 1,000 rpm on an ITC200 instrument (GE Healthcare). All data were processed using Origin 7.0 and fit to a one‐site binding model after background buffer subtraction.

The isothermal titration calorimetry (ITC) experiments were performed using an iTC200 instrument (GE healthcare, Northampton, MA, USA). ZtrLac1A (145 μM) dialyzed against 20 mM NaOAc, pH 5.5 was titrated with 2.1 mM β-cyclodextrin (β-CD) dissolved in the same buffer at 25 °C with an initial injection of 0.4 μL followed by 15 injections of 2 μL. A control titration into the dialysis buffer was used to compensate the heat of dilution. A one-binding site model was fit to the ITC data to determine the equilibrium association constant (K_a), the molar binding enthalpy (ΔH) and the stoichiometry of binding (N_o) using the ITC analysis plug in ORIGIN software provided with the instrument.

ITC experiments were conducted at 25 °C by titration of a stock sample of 100 µM compound onto a solution of 10 µM Hsp90 in a GE ITC200 instrument.