Nanoanalyze

ITC measurements were carried out on either a VP-ITC instrument (MicroCal, Northampton, MA) or an Affinity ITC instrument (TA Instruments, New Castle, DE). Protein samples were dialyzed for 3 d against 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.5 mM TCEP (ITC buffer) and either 5 mM CaCl₂ or 5 mM EGTA. The LIC1_433–458 peptide was subjected to three cycles of solubilization/lyophilization in methanol 50% (v/v) to remove any trifluoroacetic acid and acetonitrile remaining after reverse-phase purification, followed by final solubilization in ITC buffer. The concentration of the peptide was determined by fluorescence with fluorescamine-labeled LIC1_433–458. The proteins (or the LIC1_433–458 peptide) in the syringe were titrated at a concentration 10 to 20 fold higher than that of the proteins in the ITC cell of total volume 1.44 ml (VP-ITC) or 0.94 ml (Affinity ITC), as indicated in the figures. Titrations consisted of 10 μl injections, lasting for 10 s, with an interval of 300–400 s between injections. The heat of binding was corrected for the heat of injection, determined by injecting proteins into buffer. Data were analyzed using the programs Origin (OriginLab, Northampton, MA) or Nanoanalyze (TA Instruments, New Castle, DE). The temperature and parameters of the fit (stoichiometry and affinity) of each experiment are given in the figures.

The 250 µL of 10 μM DNA in 50 mM H₂KPO₄-HK₂PO₄ buffer (pH 7.0) was filled into the sample cell with a dedicated syringe, and 50 µL of 50 mM H₂KPO₄-HK₂PO₄ buffer (pH 7.0) containing 100 μM 1 solution was filled into a dropping syringe. The thermodynamic parameters were calculated by analysis in Independent mode using the data from the 2 to 25th titration, which excluded the values of the first titration. The analysis software was Nano Analyze by TA Instrument. The measurement conditions were as follows—measurement temperature: 25 °C, stirring speed: 350 rpm, cell volume: 180 μL, syringe size: 50 μL, 1–25 titrations: 1.96 μL, and interval: 120 s.

Titration experiments are performed by using a Nano ITC Low Volume calorimeter from TA Instruments (New Castle, DE, USA) in accordance with our previously adopted protocol [44 (link)]. The sample cell (700 µL) and the syringe (50 µL) are filled with aqueous solutions of HA (from 0.1 to 0.7%w/v) and Gd-DTPA (1.5 mM) respectively. Injection volumes and intervals are fixed at 1 µL and 500 s, respectively. Measurements are performed at 25 °C with a stirring rate of 200 rpm. Analysis and modeling of the raw data is carried out using the NanoAnalyze (TA instruments, New Castle, DE, USA). The function adopted to analyze the ITC data is the sum of two models: independent sites model plus a constant used for the blank (i.e., Gd-DTPA in water). The first point is excluded from the analysis. Statistics of the thermodynamic parameters are calculated on 1000 trials with a confidence level equal to 95%.