Calorimetric and Spectroscopic Studies of the Effects of the Cell Penetrating Peptide Pep‑1 and the Antimicrobial Peptide Combi‑2 on Vesicles Mimicking <i>Escherichia coli</i> Membrane

The objective of this study is to measure and compare the effects of the cell penetrating peptide (CPP) Pep-1 and the antimicrobial peptide (AMP) combi-2 on vesicles of membranes mimicking <i>Escherichia coli</i> (<i>E. coli</i>). To characterize the effects of Pep-1 and combi-2 on <i>E. coli</i> membrane vesicles, a combination of five biophysical techniques was employed: fluorescence, infrared, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Upon addition of <i>E. coli</i> membranes, tryptophan fluorescence intensity of Pep-1 showed a sudden blue-shift and decreased in a nonconcentration-dependent manner while the intensity of combi-2 decreased in a concentration-dependent manner, most significantly for a very low peptide-to-lipid ratio of 1:40. Complexes of Pep-1 and combi-2 with <i>E. coli</i> membrane mimicking vesicles having shown a significant blue-shift in fluorescence intensity were then prepared and studied in freeze-dried states. IR results indicate that Pep-1 and combi-2 adopt a major 3₁₀-helix structure in the presence of <i>E. coli</i> membrane mimicking vesicles at low peptide concentration. Pep-1 and combi-2 have a similar effect on <i>E. coli</i> membrane mimicking vesicles at low concentration even though combi-2 is in the interfacial region of the bilayer while Pep-1 is located between the interfacial region and the hydrophobic region. Combi-2 at low concentration acts as a CPP. TGA and DSC results reveal that combi-2 has a stabilizing effect on <i>E. coli</i> at any concentration while Pep-1 stabilizes the <i>E. coli</i> membrane only at high concentration. Both peptides show a preferential interaction with one of the anionic lipids leading to clustering in <i>E. coli</i> membrane. SEM images reveal that Pep-1 and combi-2 form superstructures including fibrils in the presence of <i>E. coli</i> membrane mimicking vesicles. Calorimetric and spectroscopic techniques may be used in a complementary way with imaging techniques to gain more insights into peptide–lipid interactions