# Insights into Membrane Damage by \(\alpha\)-Helical and \(\beta\)-Sheet Peptides
## 4 Conclusions
It is important to highlight that although membrane damage is a stochastic process that involves aggregation within the lipid bilayer, the results from our simulations are highly consistent. Leakage was observed in three independent simulations performed using M03 peptides, but in none of the three simulations performed with M04 peptides. Membrane damage of the latter peptides was only observed at a higher concentration, in agreement with the experiments. However, this consistency can only be obtained if simulations are performed for very long times, i.e., \(\sim\)10 us. Accordingly, the increased accessibility of computers that can reach these timescales is likely to expand the use of all-atom simulations to new interesting problems. This will require a calibration of force fields to ensure the right balance between peptide-peptide, peptide-lipid, and lipid-lipid interactions. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/biom15070973/s1](https://www.mdpi.com/article/10.3390/biom15070973/s1)]([https://www.mdpi.com/article/10.3390/biom15070973/s1](https://www.mdpi.com/article/10.3390/biom15070973/s1)), Figure S1: Molecular structure of the peptide-membrane system for the 8-residue sequences M01 and M02. Lipids are depicted in gray, and a color scheme is used to represent secondary structures. Yellow is used for \(\beta\)-strands, purple and blue for \(\alpha\)- and \(\pi\)-helices, and cyan for turn/bend; Figure S2: Molecular structure of the peptide-membrane system for the 16-residue sequences M03 and M04. Lipids are depicted in gray, and a color scheme is used to represent secondary structures. Purple and blue are used for \(\alpha\)- and \(\pi\)-helices, and cyan for turn/bend; Figure S3: Experimental leakage data of M02 with LUVs. Leakage experiments were performed at a lipid concentration of 500 \(\upmu\)M and at peptide concentrations of 100 \(\upmu\)M, 500 \(\upmu\)M, and 1 mM, corresponding to peptide:lipid ratios of 1.5, 1:1, and 2:1, respectively; Figure S4: Cryo-EM image of a sample of the LUVs produced and used in these experiments. Grids were flash frozen immediately after preparation of the vesicles and scale bar is 200 nm; Figure S5: MALDI-TOF spectra M02; Figure S6: MALDI-TOF spectra M03; Figure S7: MALDI-TOF spectra M04; Figure S8: Analytical HPLC trace M02; Figure S9: Analytical HPLC trace of M03; Figure S10: Analytical HPLC trace of M04; Figure S11: Representative analytical HPLC concentration curve of M02; Figure S12: Representative analytical HPLC concentration curve for M03 and M04; Figure S13: Representative phosphorus concentration curve; Table S1: Preparatory scale HPLC gradients for purification; Table S2: Calculated and observed m/z for all peptides by MALDI-TOF-MS; Table S3: Analytical HPLC gradient conditions and peptide retention times. **Author Contributions:** Conceptualization, W.R., H.E.D., B.L.N. and C.L.D. ; Investigation, W.R. and H.E.D. ; Resources, B.L.N. and C.L.D. ; Writing--original draft, W.R., H.E.D. and C.L.D. ; Writing--review & editing, B.L.N. and C.L.D. ; Visualization, W.R., H.E.D. and C.L.D. ; Project administration, B.L.N. and C.L.D. ; Funding acquisition, B.L.N. and C.L.D. All authors have read and agreed to the published version of the manuscript. **Funding:** WR, HED, BLN, and CLD are supported by the National Science Foundation under Grant No. CHE-2304852 and CHE-2304853. Computational resources were provided by the Academic and Research Computing System (ARCS) at New Jersey Institute of Technology. Electron microscopy was performed in the Electron Microscopy Resource at the Center for Advanced Research Technologies at the University of Rochester Medical Center. The research reported in this publication was supported by the Office Of The Director, National Institutes Of Health under Award Number S10 OD030302. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.