Peaq itc calorimeter

ITC experiments were carried out at 25 ± 0.2 °C by a MicroCal PEAQ-ITC calorimeter. Arsenic binding flagellin variants in monomeric form were prepared in 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) ((Sigma Aldrich), 150 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich) (pH 7.0). Before the measurement 10% v/v DMSO was added to the solution. As a titrant, phenylarsine oxide (PAO) (Sigma Aldrich) was first dissolved in pure dimethyl sulfoxide (DMSO) (Sigma Aldrich) to an appropriate concentration then diluted 10 times with 100 mM HEPES, 150 mM NaCl (pH 7.0) buffer containing 1 mM TCEP. A typical experiment consisted of a ~ 0.5 mM PAO solution titrated into a ~ 5 µM protein solution in the same buffer. Interaction between PAO and the buffer or the buffer and protein was checked by PAO-to-buffer and buffer-to-protein titrations, respectively. The ITC data were fitted to a one-binding-site model with the MicroCal PEAQ-ITC Analysis Software package provided by MicroCal, using non-linear least-squares algorithm.

Calorimetric titrations were carried out on a MicroCal PEAQ-ITC calorimeter (Malvern, UK) at 25°C. Before titrations, all KIT mutant proteins used were buffer-exchanged into an identical lot of HBS buffer (10 mM Hepes pH7.5, 150 mM NaCl). Gel filtration was used to control buffer heat dilution effects. Each protein sample was thoroughly degassed before ITC experiments, and these were run in triplicate (see Supporting information appendix for details).

ITC determined the observed standard enthalpy changes upon binding as well as the K_{d_obs} values [41 (link)] that were compared to ones obtained by FTSA. The ITC experiments were performed by adding the proteins to the calorimeter cell and the compound solutions to the syringe at 37 ºC. The CAII binding with compounds 28, 31, and 36 and CAVB with compound 32 (Figure S104) were determined using a MicroCal PEAQ-ITC calorimeter (Northampton, MA, USA). Experiments consisted of 19 injections, the first injection of 0.4 μL over 0.8 s, with the remaining injections of 2 μL over 4 s with spacing of 150 s between injections, at temperature 37 °C, reference power 10 μcal/s, and stirring speed of 750 rpm. The protein concentrations for CAII and CAVB were 10 μM and 8 μM, respectively; the compound concentration was 10 times higher than the protein (100 μM for compounds tested with CAII, 80 μM for compound tested with CAVB). Proteins and compounds were diluted in sodium phosphate 50 mM buffer at pH 7.0, containing 100 mM NaCl and 2% (v/v) DMSO. Data were analyzed using NITPIC (version 1.2.7) [49 (link),50 (link)] and Sedphat (v. 14.0) [51 (link)] software; the heat of dilution was subtracted from the titration curves.

All ITC experiments were performed on a MicroCal PEAQ-ITC calorimeter at 20 °C. Prior to titration, 0.05-0.1 mM Bifidobacterium SAM-VI riboswitch RNA was dialysed overnight at room temperature against ITC buffer containing 40 mM HEPES, pH 7.0, 50 mM KCl, 10 mM MgCl₂. RNAs were refolded at 65 °C for 5 min and cooled on ice before titration. The ligands were dissolved in the dialysis buffer at the concentration of 0.5-1 mM and injected into the sample cell that was filled with 203 μl of RNA sample in a volume of a single initial injection of 1 µl, followed by 18 injections of 2 µl ligand into RNA sample, with a 0.5 μl s⁻¹ rate, 120 s intervals between injections and a reference power of 5 μcal s⁻¹. Integrated heat data were analyzed using a one-site binding model via MicroCal PEAQ-ITC Analysis Software, provided by the manufacturer. All ITC titration experiments were independently repeated (three replicates in total) and the complete sets of thermodynamic binding parameters are provided in Supplementary Table 2.

Titrations were performed under standard conditions, as applied previously, to ensure comparability (Kutzner et al. 2019 (link); Ludwig et al. 2019a (link)). Removal of cognate sugar from protein preparations was guaranteed by dialysis over the course of 72 h with five buffer exchanges (2 L each). Briefly, 2 µL aliquots of ligand-containing solution (from a total of 36.4 µL) were injected per step into the galectin-containing solution in 20 mM phosphate buffer at pH 7.2 with 10 mM NaCl and 10 mM β-mercaptoethanol, starting at a volume of 200 µL. Injections were performed every 180 s at 25 °C and 500 rpm in a PEAQ-ITC calorimeter (Malvern, Westborough, MA, USA). Protein concentrations were based on absorbance applying a sequence-based extinction coefficient calculated with ExPASy ProtParam software. Data were routinely processed by MicroCal PEAQ-ITC analysis software with the one-set-of-sites/sequential models and a fitted offset parameter to account for potential background signal.