Microcal itc200

To examine the binding affinity and specificity of the selected aptamer, we used isothermal titration calorimetry (ITC) MicroCal iTC200 (Malvern Panalytical, Malvern, UK) [27 (link)]. AFB1, AFB2, AFG1, AFG2, OTA, and FB1 were dissolved in DMSO and added to the buffer. The thermal equilibration was set at 25 °C with an initial 60-s delay step with 125 μM AFB1, AFB2, AFG1, AFG2, OTA, or FB1 in the syringe and 5 μM selected ssDNA aptamer in the sample cell dissolved in buffer (0.1 M Tris-HCl pH 7.4 in 1% DMSO). The binding experiment involved using a 0.4-μL injection at the first injection, followed by a 17-consecutive 2.1-μL injection every 60 s. The reference power setting was 5 mcal/s, and the syringe stirring speed setting 800 rpm. The control was set up under the same conditions as in the binding experiment by injecting the toxins without the addition of DNA samples. Calorimeter software was used to analyze the data.

The binding affinities of DENV-LP to heparin were determined using isothermal titration calorimetry (ITC) (MicroCal iTC200, Malvern Panalytical Ltd, Enigma Business Park, UK). Titrations were performed at a temperature of 25 °C by injecting 2 μl aliquots of 1000 μM ligand dissolved in 1 × PBS buffer into a cell containing 10 μM DENV-LP. The heat release was recorded, and the titration data were analyzed with MicroCal Origin ITC software (Malvern Panalytical Ltd). Thermodynamic parameters were determined by fitting experimental data with nonlinear least-squares using the one-set sites binding model (Duff et al. 2011 (link); Ikegaya et al. 2021 (link)).

SLC39A7 peptides that contain either no (Me0; HGHSHAHGHGHTH) or six 1MH (Me6, HGXSXAXGXGXTX; X = 1MH)) residues were synthesized with a peptide synthesizer (ABI, 433 A) using Fmoc-3-methyl-L-histidine (Merck) for the 1MH residue. The peptides were purified with HPLC, and purity determined by MALDI-MS. Dry weights were quantified with an amino acid analyzer (Hitachi, L-8900) for the calculation of molarity. ITC assay was performed as reported previously^{51 (link)}, with a slight modification. Briefly, SLC39A7 peptides and Zn²⁺ solutions were prepared in 20 mM Tris –HCl pH 7.5, 100 mM NaCl. Titration of ZnCl₂ (2 mM) into peptide solutions (50 μM) was performed at 30 °C using an ITC titration calorimeter (Malvern Panalytical, MicroCal iTC200). 39 injections of 1 µL were made with an equilibration time of 1 min between injections. Integration of the thermograms after correction for heats of dilution yielded binding isotherms that fit best to a one-set of sites binding model using the ITC data analysis software Origin 7.0 (MicroCal Inc., Piscataway, NJ). A non-linear least-squares curve-fitting algorithm was used to determine the stoichiometric ratio (n), the dissociation constant (K_d), and the change in enthalpy (ΔH) of the interaction. All ITC experiments were performed in triplicate.

ITC experiments were performed using a MicroCal iTC200 (Malvern Instruments) using either chelex-filtered HBS (25 mM Hepes, 150 mM NaCl, pH 7) or low-salt MES buffer (25 mM MES, 40 mM NaCl, pH 6). The titrant was dissolved in the same buffer as was used for dialysis of the protein sample. Using the iTC200, the titrant CaCl₂ (15 mM stock) was added in defined steps of 1 to 2.5 μl to 80 μl protein solution at 298 K while stirring at 750 rpm. The differential heat of each injection was measured and plotted against the molar ratio. The data were fitted to a one-set of sites binding model assuming a Hill coefficient of 1. Owing to the low c-values of the measurements (c < 5), the enthalpy could not be determined reliably. See also Figs. S29 and S30.

Titrations were performed using the MicroCal iTC200 and MicroCal Auto-iTC200 instruments (Malvern Panalytical, Malvern, UK). Binding assays were carried out in 150 mM NaCl, 50 mM Tris pH 8, 1 mM DTT and 0–1% v/v DMSO. Generally, 13–20 injections were carried out with a DP of 6 μcal/s, 750 RPM, 20–25 °C, 150–180 s spacing and 5–6 s injection times of 2.5–3 μL. Titrations were performed with protein and peptide in cell and syringe interchangeably. Generally, TRIM7 in the cell was kept at 20 μM with peptides diluted to 400 μM and up to 2 mM for dipeptides. See Table S2 for details. Control titrations of titrant into buffer were carried out where appropriate. Data were analyzed using MicroCal PEAQ-ITC analysis software, using one site model to fit the data. For low c-value experiments (such as the LQ titration), the N was fixed to 1. For experiments with MBP-T7CCPS, the buffer was 165 mM NaCl, 50 mM Tris pH 8, 1 mM DTT, 0.5% w/v glycerol and 0–0.8% v/v DMSO. For experiments with rbGYG, mGYG and mTrim7-PRYPSRY, the protein samples were dialysed against 50 mM phosphate buffer, pH 6.5 and 1 mM DTT. ITC experiments were conducted on MicroCal ITC 200 at 15 °C or an AutoITC and analysed using a standard one-state model within MicroCal instrument software.

Experiments were carried out on a MicroCAl iTC200 (Malvern Instruments) at 15°C in 20 mM potassium phosphate (pH 7.0), 150 mM NaCl, and 1 mM β-mercaptoethanol. In all cases concentrated Nab3_191-261 (198 μM) in the syringe, was titrated into Nrd1 variants: Nrd1_{147-222/290-489} (19 μM), Nrd1_1-222 (28 μM), and txAHTEV-Nrd1_147-222 (54 μM). Experiments were performed in duplicate with injections of 2 μl (0.4 μl for first point) separated by 150 s delays to recover thermal power baseline and continuous stirring in the cell (1,000 rpm) for correct mixing. The reference cell was filled with water in all the experiments. Data were processed by removing the blank experiment (dilution of Nab3_191-261 in buffer) and adjusted to one-site binding model with Origin 7.0 (OriginLab).