Low volume nanoitc

LUVs containing Lipid II (2%, mol/mol) were prepared by mixing the appropriate volumes of Lipid II and DOPC stock solutions in CHCl₃/MeOH (2:1, v/v). The lipid solutions were dried by a nitrogen stream and hydrated with 10 mm Tris-HCl, 100 mm NaCl, and pH 8 buffer to a lipid-phosphate concentration of ~ 20 mm. LUVs were obtained after 10 times freeze-thaw cycles followed by 10 rounds of extrusion through 200 nm membrane filters (Whatman Nuclepore, Track-Etch Membranes). The concentration of lipid-phosphate was determined as described (Rouser et al., 1970 (link)).
Isothermal titration calorimetry was performed with the Low Volume Nano ITC (Waters LLC, New Castle, DE, United States) to determine the interaction between LUVs and brevibacillins. Brevibacillins were diluted in a buffer (10 mm Tris-HCl, 100 mm NaCl, and pH 8) to a final concentration of 50 μm. Samples were degassed before use. The chamber was filled with 177 μl of the brevibacillins solutions, and the LUVs were titrated into the chamber at a rate of 2 μl/300 s with a stirring rate of 300 rpm. Experiments were performed at 25°C. Control experiments were performed with Lipid II-free LUVs. The Kd values of brevibacillins to Lipid II were calculated using the Nano Analyze Software (Waters LLC).

For ITC analysis, vesicles of POPC and POPG were generated using the extrusion technique as previously reported²⁵ . Phospholipid content was determined as inorganic phosphate after treatment with perchloric acid by UV-VIS spectroscopy^{26 (link)}. POPC and 10% POPG/90% POPC vesicles were diluted to 1.5 mg/mL, while POPG vesicles were further diluted to 0.15 mg/mL. For measurements using BLES, the stock solution was diluted to 1.5 mg/mL. Interactions between CATH-2 and large unilamellar vesicles consisting of POPC and/or POPG, or between CATH-2 and BLES were tested using ITC. All ITC experiments were performed on a Low Volume NanoITC (TA instruments - Waters LLC, New Castle, USA). In each experiment, the ITC cell chamber was filled with 190 µl of vesicles or BLES, and the syringe was filled with a 50 µl solution of 320 µM CATH-2. Titrations were incremental with 2 µl injections at 300 seconds intervals. Experiments were performed at 37 °C and data were analyzed with the Nano Analyze software (TA instruments - Waters LLC).