# Insights into Membrane Damage by \(\alpha\)-Helical and \(\beta\)-Sheet Peptides
## 3 Results
### Experimental Investigation
#### 3.3.1 Secondary Structure  
To investigate the secondary structure of the peptides modeled above, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra of each peptide sequence were obtained and analyzed. Both CD and FT-IR spectra were recorded immediately after dissolving the peptides in the liposome elution buffer. For the experimental details, please refer to the Methodology section. For CD, additional spectra were recorded in the presence of large unilamellar vesicles (LUVs). The CD spectra (Figure 5A,B) indicate that both peptides adopted at least a partial \(\alpha\)-helical secondary structure, as evidenced by minima at 208 nm and 222 nm [70]. Peptide M03 also exhibited a positive peak at 190 nm, another characteristic feature of an \(\alpha\)-helix. However, in addition to the typical \(\alpha\)-helical peaks, both peptides showed spectral features consistent with \(\beta\)-sheet structures, such as minima at 218 nm. M04 also displayed a maximum at 197 nm, indicative of a \(\beta\)-sheet character. These results suggest that the secondary structure of each peptide was a complex mixture of \(\alpha\)-helical and \(\beta\)-sheet content. Additionally, the spectra recorded in the presence of the LUVs differed from those recorded in buffer alone. Peptide M03 showed an increase in CD signal as the peptide concentration increased from 100 uM to 200 uM in the absence of LUVs (Figure 5A). However, spectra recorded in the presence of lipid vesicles showed a marked decrease in signal at both concentrations, likely due to aggregation of the peptides as they interacted with the lipid membrane. At peptide concentrations higher than 500 uM, M03 began to lose solubility, while M04 began to precipitate from solution at concentrations higher than 200 uM. The reduced solubility of M04 was likely due to the orientation of all its hydrophobic residues on one face of the \(\alpha\)-helix, making it more prone to aggregation in aqueous solutions. Interestingly, M04 in the absence of liposomes also showed an increase in CD signal at 200 uM compared to 100 uM. At a concentration of 100 uM, the  
Figure 5: CD spectra in liposome elution buffer of (**A**) M03 and (**B**) M04 at various peptide concentrations, with and without liposomes. Lipid concentration was 500 uM for all experiments containing liposomes. (**C**) FT-IR of M03 and M04 at 500 uM. CD signal was stronger in the presence of the liposomes than in their absence, suggesting that interaction with the lipid membrane may have induced the formation of a secondary structure in M04, a common characteristic of membrane-active peptides [71; 72; 73]. It is also important to note that the CD spectra of M03 and M04 were noticeably different from each other. This underscores the effect that varying the sequence pattern, even slightly, can have on the resulting peptide properties. While CD spectroscopy is a classic technique for probing secondary structures, its analysis can become complicated, especially in peptides and proteins containing aromatic residues [70]. Therefore, Fourier transform infrared (FT-IR) spectroscopy can also provide valuable insights into peptide secondary structure by analyzing shifts in the absorbance of the amide I stretch. Peptides with \(\alpha\)-helical structures typically exhibit absorbance between 1655 and 1638 cm\({}^{-1}\), while those with \(\beta\)-sheet character absorb around 1630 cm\({}^{-1}\) in D\({}_{2}\)O [74]. Unordered structures are indicated by absorbance at approximately 1650 cm\({}^{-1}\). Both peptides M03 and M04 displayed peaks near 1650 cm\({}^{-1}\) and 1630 cm\({}^{-1}\) (Figure 5C). While the peak at 1650 cm\({}^{-1}\) could represent either disordered or \(\alpha\)-helical structures, the FT-IR spectra, in conjunction with the CD results, suggest an \(\alpha\)-helical orientation is more likely. However, the presence of \(\beta\)-sheet indicators in both CD and FT-IR spectra suggests that the peptides adopt a mixture of \(\alpha\)-helical and \(\beta\)-sheet structures in solution.