Itc200 isothermal titration calorimeter

The enzyme solutions (70 μM) were loaded into an iTC₂₀₀ isothermal titration calorimeter (Microcal, Inc.), which has an active cell volume of 280 μl. The solution was titrated with 20 injections of a 10 mM EA solution at 3-minute intervals using a 2-μl titration syringe. The stirring speed was set to 1000 rpm. The experiments were conducted at a constant temperature of approximately 25°C. The ligand solutions were prepared in the same buffer (HEPES, pH 7.4) as the protein solutions. The titration experiments were repeated three times. The raw data were corrected for dilution effects, and the concentration was normalized prior to data analysis using the “ONESites” model of the MicroCal version of ORIGIN 7.0. During the fitting process, K_A (association constant) and ΔH were allowed to float.

ITC was used to measure the GDP-binding affinities of wild-type EcNFeoB and mutant proteins. Protein (approximately 0.1 mM) in buffer (20 mM Tris pH 8.0 and 100 mM NaCl) was equilibrated for 1 min at 25 °C with stirring (1000 rpm) in the sample cell of a MicroCal iTC₂₀₀ Isothermal Titration Calorimeter. GDP (2.5–5 mM) was titrated into the sample cell in 0.5–2 μl injections over 0.8 s with 150 s spacing between injections. Power input (μcal s⁻¹) required to maintain equal temperatures between the sample and reference cells in response to each addition of ligand was plotted versus time (min). The data were integrated and plotted versus the molar ratio of ligand to protein. Non-linear regression was used to obtain the thermodynamic parameters (including GDP-binding affinity, K_a). Data were fitted to a one-site binding model using the Origin 7 Software (MicroCal) to obtain stoichiometry (N), enthalpy (ΔH), entropy (ΔS) and association rate constant (K_a). The dissociation constant (K_d) was calculated from Equation 1 (K_d=1/K_a). All reported values are the average of three or more independent titrations. Due to interdiffusion of the solutions during the insertion of the syringe into the sample chamber, the first injection is not useful for analysis and was omitted from all calculations.

The binding of ions to the S100A1 protein was studied on an iTC200 isothermal titration calorimeter (MicroCal, Los Angeles, CA, USA) at 37 °C, in 50 mM Tris buffer and 1 mM tris(2-carboxyethyl)phosphine (TCEP) at pH 7.5, as described previously [20 (link),30 (link)]. The protein concentration in the calorimetric cell was 50 µM whereas the concentration of zinc and calcium ions in the titrating syringe was 0.5 mM. The volume of the injections was 0.5 µL before the ion concentration reached the equimolar ratio with protein, and then set to 2.5 µL. The dilution heat was measured by titration of the ion-containing buffer into the same buffer without the protein. The obtained curve was analyzed by MicroCal Origin 7.0 software (Los Angeles, CA, USA) using the models of the two types of binding sites. Therefore, the stoichiometry (N), constant (K_a), enthalpy (ΔH), and entropy of binding (ΔS) were calculated from the standard thermodynamic equations.

ITC experiments were carried out on an ITC200 isothermal titration calorimeter (Microcal) at 20° C. Protein pairs used in the binding analyses were dialyzed against the same buffer (10 mM Tris/HCl pH 8, 150 mM NaCl) to minimize undesirable buffer-related effects. The dialysis buffer was used in all preliminary equilibration and washing steps.
The concentrations of purified wt and mutated N_TAIL proteins in the microcalorimeter cell (0.2 mL) ranged from 25 μM to 180 μM. XD was added from a computer-controlled 40-μL microsyringe via a total of 19 injections of 2 μL each at intervals of 180 s. Its concentration in the microsyringe ranged from 300 μM to 960 μM.
A theoretical titration curve was fitted to the experimental data using the ORIGIN software (Microcal). This software uses the relationship between the heat generated by each injection and ΔH° (enthalpy change in kcal mole^-1), K_A (association binding constant in M^-1), n (number of binding sites per monomer), total protein concentration and free and total ligand concentrations. The variation in the entropy (ΔS° in cal mol^-1 deg^-1) of each binding reaction was inferred from the variation in the free energy (ΔG°), where this latter was calculated from the following relation: ΔG° = -RT ln 1/K_A.

Enthalpy values were measured by using an iTC₂₀₀ isothermal titration calorimeter (Microcal, Milton Keynes, United Kingdom). Gyrase subunits and domains (GyrA55, GyrB43, and GyrB47) were dialyzed extensively against binding buffer [50 mM Tris–HCl (pH 7.5), 1 mM EDTA, and 100 mM KCl]. During the titration, the protein (200 μl, at 5–15 μM) was added to the sample cell, and 27 successive aliquots of SD8 (at 100–250 μM) [1.5 μl volumes except for the first one (0.5 μl)] were injected at 2-min intervals. All titrations were carried out at 25 °C. A constant DMSO concentration of 3% (v/v) (obtained by addition of DMSO to protein and SD8 samples) was used to aid SD8 solubility. The upper limit of SD8 concentration was 250 μM due to the poor solubility of the drug at higher concentrations. Data were analyzed by nonlinear regression using a single-site binding model with Origin software (Microcal), which yielded independent values for K_d.

ITC titrations were performed with a MicroCal™ iTC200 Isothermal Titration Calorimeter (Malvern) at 25°C. Protein and DNA absorbance were measured after dialysis by NanoDrop (NanoDrop Technologies) and their concentrations were determined with their respective extinction coefficients. Twenty-five 1.5 μl injections of each DNA were titrated into LANA_DBD protein solution. Data were corrected for nonspecific heats and analysed using MicroCal Origin^® 7.0 software using a one-site binding model. CHASM software was used for fitting and calculating thermodynamics parameters for a two-site binding model (49 (link)). The experiments were performed in triplicate and showed similar results. Proteins were freshly prepared for the ITC experiments and used within 24 h after the purification from the gel-filtration column, which is crucial due to the kLANA DBD's propensity to form higher oligomers. The annealed dsDNA oligonucleotides were dialysed overnight at 4°C using Slide-A-Lyzer^® Mini dialysis unit (Thermo Scientific) against buffer D (25 mM Na/K phosphate, 250 mM NaCl, 5% glycerol pH 7.0 (25°C)).