SEC-purified Bt1762-63-SusCD complex was dialysed overnight at 4 °C with 50 kDa MWCO dialysis tubing into 100 mM HEPES, pH 7.5, containing 0.05% LDAO. The dialysis buffer was used to resuspend SEC-purified FOS fractions produced by partial digestion of Erwinia levan. ITC was performed using a MicroCal PEAQ-ITC machine with v1.21 control software for data collection (Malvern Panalytical). Briefly, pure SusCD complex (25–50 µM; concentration determined by A280) in a 200 µl reaction well was injected 20 times with 2 µl aliquots of FOS (0.25–4.5 mg/ml) at 25 °C. Integrated heats were fit to a one set of sites model using the Microcal PEAQ-ITC analysis software v1.30 (Malvern Panalytical) to obtain Kd, DH and N (number of binding sites on the protein). For most titrations, the molar concentration of ligand used for the fits was based on the DP of the major FOS species present as determined by the TLC and MS analysis. For fractions 118–115, the concentration of ligand was varied such that N = 1.
Microcal peaq itc analysis software v1
Manufactured by Malvern Panalytical
Microcal PEAQ-ITC analysis software v1.30 is a tool for analyzing data from Isothermal Titration Calorimetry (ITC) experiments. It provides advanced data processing and analysis capabilities to researchers working with Malvern Panalytical's ITC instrumentation.
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4 protocols using microcal peaq itc analysis software v1
1
Binding Affinity of Bt1762-63-SusCD Complex
2
Quantifying CBS-SAM Binding Kinetics
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Purified CBS proteins were buffer exchanged into 20 mM HEPES, pH 7.4 using Zeba spin columns (Thermo Fisher Scientific) at 4 °C. To prevent precipitation CBSFL-CHis and its mutants were buffer exchanged into 20 mM HEPES, pH 7.4, 0.01% triton X-100 whereas CBSRD was exchanged into 20 mM HEPES, pH 7.4, 200 mM NaCl. The appropriate buffer was then used to dissolve SAM from stocks to 500 µM. A MicroCal PEAQ-ITC machine with v1.21 control software for data collection (Malvern Panalytical) was used to perform ITC. CBS constructs were tested at 30-60 µM monomer in a 200 µl sample cell and were injected with 0.4 µl followed by 44 × 0.8 µl of SAM with 150 s spacing at 25 °C. Heats of dilution were determined by separate runs of SAM injected into the buffer alone. Integrated heats were fitted using the Microcal PEAQ-ITC analysis software v1.30 (Malvern Panalytical) to obtain n, Kd, ∆H, and −T∆S.
3
ITC Analysis of MavL-ADPR/Ub Interactions
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The ITC experiments measuring the Kd were performed at 25 °C using MicroCal PEAQ-ITC (Malvern Panalytical). Specifically, 200 μM MavL42-435 was titrated with 2 mM ADPR or 2 mM Ub (or Ub mutant) in the buffer containing 25 mM HEPES 7.4, 100 mM NaCl, and 1 mM TCEP. Raw data were integrated and analyzed by MicroCal PEAQ-ITC Analysis Software v1.41 (Malvern Panalytical) with a one-binding site model to determine the Kd values.
4
Calorimetric Characterization of CdaA Ligand Binding
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ITC experiments were performed at 25°C and a stirring speed of 524 rpm on a MicroCal VP-ITC microcalorimeter (MicroCal Inc). Measurements were carried out with 50 μM CdaA in the sample cell and 1 mM of the analyzed ligand in the titration syringe (compounds 7 and 4, ATP, and c-di-AMP). Both, protein and ligands, were dissolved in the same buffer composed of 20 mM Tris-HCl pH 7.5, 300 mM NaCl. In case of compounds 7 and 4, the buffer has been supplemented with 2% DMSO, whereas 10 mM MgCl2 has been added in case of ATP and c-di-AMP. In the presence of Mg2+-ions, the metal-dependent Lm CdaA was shown to be not catalytically active (Heidemann et al. 2019 (link)). Furthermore, the common control experiments have been carried out: titrant to buffer, buffer to protein, and finally buffer to buffer. For compound 7, ATP, and c-di-AMP, the titrant to buffer experiments showed significant signals, which were considered in the subsequent analysis. Data was analyzed using the MicroCal PEAQ-ITC Analysis Software v1.41 (Malvern Panalytical) employing the single control method (subtraction of titrant to buffer experiment). For all performed experiments, the data sets were fit with a 1:1 binding model and yielded an assessment of the following thermodynamic parameters: dissociation constant (KD), a stoichiometry N, and the enthalpy of interaction ΔH.
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