Microcal vp itc microcalorimeter

All ITC runs were performed at 25 °C on a MicroCal VP-ITC microcalorimeter (Malvern, Westborough, MA). 200–400 μM peptide was titrated into 8 μM SakΔN10 or Sak in 20 mM HEPES, pH 7.4. In preliminary trials, the inclusion of salt (100 mM NaCl) in the ITC buffer was found to promote peptide aggregation in the injection syringe as indicated by endothermic heats of dilution in peptide-to-buffer control experiments. Data analysis was performed using the MicroCal Origin software using the “One Set of Sites” binding model. At least two experiments were performed for each titration. Statistical analysis was performed to confirm the statistical validity of the differences in the K_D values determined for LfcinB and Tritrp binding to SakΔN10 or Sak. p-values were calculated from Student’s t-tests at the 95% confidence level with a one-tailed hypothesis.

The electronic absorption spectra (UV/Vis) were recorded on a Varian Cary 100 Bio spectrophotometer (Agilent, Santa Clara, CA, United Staets) and circular dichroism (CD) spectra on a JASCO J815 spectrophotometer (ABL&E Handels GmbH, Wien, Austria) at 25°C using appropriate 1 cm path quartz cuvettes (Eriksson and Nordén, 2001 (link)). The calf thymus DNA (ctDNA) was purchased from Sigma-Aldrich. Isothermal titration calorimetry (ITC) experiments were performed on a MicroCal VP-ITC microcalorimeter (MicroCal, Inc., Northampton, MA, United States) (Chaires, 2006 (link)). Origin 7.0 software, supplied by the manufacturer was used for data analysis. All additional data of these experiments are provided in the Supplementary Materials.

Isothermal titration calorimetry (ITC) experiments were performed on a Microcal VP-ITC microcalorimeter (MicroCal, Northampton, MA). In a typical experiment with the mGRFT tandemers and monomeric, glycosylated, bacculovirus-produced HIV-1_IIIB gp120 (Immunodiagnostics, Inc., Woburn, MA), the mGRFT tandemer protein (180 μM) was placed in the syringe injector and the gp120 was placed in the calorimeter cell (2.5 μM). In all experiments, a total of 55 injections of tandemer (5 μl/injection) were made, with 600 s spacing between injections. The titrations were all done in a rapidly stirring solution (300 rpm) held at a constant temperature of 30°C. The heats of binding were recorded as the excess power compensation required for maintaining the same temperature during the course of the titration. Baseline experiments of tandemer titration into buffer were done to calculate heats of dilution and this value was subtracted from the experimental heats of binding. The resulting isotherms were fitted using Origin 5.0 nonlinear least-squares program according to manufacturer’s protocol, and the values for the enthalpy of binding (ΔH) and the dissociation constant were obtained. From the dissociation constant, a value for the free energy of binding (ΔG) was extrapolated (ΔG = −RTlnKa), and from this value, the entropy of binding (ΔS) was lastly calculated (ΔG = ΔH –TΔS).

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 (K_D), a stoichiometry N, and the enthalpy of interaction ΔH.