Isothermal Titration Calorimetry

The interaction between the peptides and LUVs was measured by isothermal titration calorimetry (ITC) as described before (Alegre-Cebollada et al., 2008; Maula et al., 2013; García-Linares et al., 2014; Rivera-De-Torre et al., 2017), using a VP-ITC calorimeter (Malvern MicroCal Worcestershire, United Kingdom). Briefly, 20.0–80.0 μM peptide solutions were titrated by injection of 20 μL aliquots of lipid suspensions (phospholipid concentration of 10.0 mM) at a constant temperature of 25°C. The buffer employed was 50 mM sodium phosphate, pH 7.0. Binding isotherms were adjusted using the standard MicroCal software with the OneSites model available within the Origin program where the “n” value refers to an average mean value of lipids affected by peptide binding to the bilayer. This does not necessarily imply specific direct contact between all n lipid molecules and the peptide. The c values (c = K_a × P₀) for all the graphs were in the range 1–1000, the needed requirement in order to produce thermograms with the curvature required for the simultaneous determination of K_a and ΔH (Wiseman et al., 1989; Alegre-Cebollada et al., 2008). K_a is the affinity constant calculated according to the mentioned software model and P₀ is the peptide concentration employed in the experiment.