Selective Membrane Disruption Mechanism of an Antibacterial γ-AApeptide Defined by EPR Spectroscopy  Pavanjeet Kaur, Yaqiong Li, Jianfeng Cai, Likai Song  Biophysical Journal  Volume 110, Issue 8, Pages 1789-1799 (April 2016) DOI: 10.1016/j.bpj.2016.02.038 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Structure of lipo-cyclic-γ-AApeptide 1 (A) and comparison of an α-peptide (B) and a γ-AApeptide (C). A γ-AApeptide is comparable to an α-peptide in unit length and half of the side chains of a γ-AApeptide are linked to the amide groups. The lipo-cyclic-γ-AApeptide 1 contains a cyclic γ-AApeptide and a lipid tail. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Membrane permeability changes induced by AA1 binding. (A) Comparison of the permeability changes of liposomes with different lipid compositions at an L/P of 10. The following lipids were compared: POPC/POPG, POPC, and POPC with 10, 30, and 50% CHOL. (B) Permeability changes of POPC/POPG liposomes with L/Ps ranging from 10 to 80. Note: A 100% permeability change indicates complete 4-PT and VC mixing and 4-PT signal reduction due to membrane penetration. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Membrane fluidity changes in the presence of AA1. Room temperature EPR spectra and mobility changes upon AA1 binding of liposomes labeled with 5-SASL for (A) POPC/POPG, (B) POPC, and (C) POPC/30% CHOL liposomes at an L/P of 10. EPR spectra of bare liposomes are overlaid with the spectra in the presence of AA1. The 2T|| changes (Δ2T||) on AA1 binding are shown on the upper-right side of the spectra. To see this figure in color, go online. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Lipid lateral ordering. 94 GHz EPR spectra of POPC/POPG (A) and POPC (B) liposomes with 5-SASL in the absence and presence of AA1. Lateral ordering was detected for (A) with arrows showing the 〈gxx〉, 〈gyy〉, and 〈gzz〉 components. The L/P is 10. No sign of lateral ordering was observed for (B). (C) Membrane structure showing transverse and lateral order and the corresponding g-factor anisotropy, indicated by gxx, gyy, and gzz. The averaged principle axes of a nitroxide spin label (5-SASL) aligned with regard to a bilayer are shown. Changes in lipid lateral order are reflected in the x and y components of the g tensors of the spin label. To see this figure in color, go online. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Accessibility changes upon the binding of AA1. Changes in O2 (A) and NiEDDA (B) accessibility of POPC/POPG liposomes with 5-SASL on the addition of AA1 are shown. (C) Comparison of the percentage of the O2 accessibility changes of POPC/POPG, POPC, and POPC/30% CHOL liposomes upon AA1 binding. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Depth parameter changes of 7-PC, 10-PC, and 12-PC upon AA1 binding to POPC/POPG liposomes when compared to bare liposomes. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Membrane interaction and disruption mechanism of AA1. (A) AA1 binds to the membrane through electrostatic and hydrophobic interactions. (B) Insertion of the bulky hydrophobic groups of AA1 into the membrane results in lateral expansion of the upper leaflet of a lipid bilayer. (C) Lipid lateral expansion leads to membrane thinning. Moreover, peptide insertion causes transient membrane disruption or local bilayer disorder, subsequently leading to permeability changes. To see this figure in color, go online. Biophysical Journal 2016 110, 1789-1799DOI: (10.1016/j.bpj.2016.02.038) Copyright © 2016 Biophysical Society Terms and Conditions