BIOPHYSICAL CHARACTERIZATION OF Ctx(Ile21)-Ha ANTIMICROBIAL PEPTIDE AND ITS ANALOGUES LABELED WITH TOAC SPIN LABEL: INTERACTION WITH MEMBRANE MODELS IS DRIVEN BY FLUIDITY AND LIPID MEMBRANE CHARGES

Antimicrobial peptides (AMPs) permeabilize bacterial membranes, leading to cell death, and are promising candidates for new antibiotics development. Specifically, the antimicrobial peptide Ctx(Ile²¹)-Ha showed strong antibacterial and antifungal activity with moderate human cell toxicity. This study aims to elucidate the detailed membrane interactions between Ctx(Ile²¹)-Ha and its TOAC-labeled analogues with large unilamellar vesicles (LUVs) by Langmuir monolayer technique to investigate the peptide surface activity and its affinity and interaction at physiological pH. In addition, dynamic light scattering (DLS), Zeta potential (ζ), circular dichroism (CD), and electron spin resonance (ESR) assays were conducted using several membrane mimetics, POPC, POPC/CHOL, POPC/ERG, POPC/CHOL/POPG, POPC/POPG, POPC/POPS, and POPE/POPG, to mimic human, bacterial, and fungal membranes. Gibbs adsorption isotherms demonstrated that the peptide was able to adsorb at the air–water interface, forming a stable interfacial film. Insertion assays performed at constant areas, under conditions simulating the lipid packing density of biological membranes, revealed that Ctx(Ile²¹)-Ha is capable of distinguishing between phosphatidylserine (PS) and phosphatidylglycerol (PG), based on their distinct packing perturbation profiles. The incorporation of sterol in PC/sterol mixtures attenuated the peptide-induced packing disruption, suggesting a stabilizing effect against membrane perturbation. Conversely, substitution of the zwitterionic lipid by sterol in PG/sterol mixtures did not mitigate the perturbative effect of the peptide, underscoring the combined influence of membrane fluidity and surface charge on peptide–membrane interactions. CD analyses revealed that the peptides adopt an α-helical structure upon binding to the selected membranes. Membrane-binding partition constant (K_P) obtained from peptide titration via CD, demonstrated higher affinity for sterol-containing membranes than for anionic ones. ESR spectroscopy, combined with nonlinear least squares (NLLS) simulations, provided complementary insight into peptide–membrane interactions. As membrane concentration increased, ESR spectra showed the progressive emergence of a second spectral component associated with membrane-bound peptide. Partition constants Kp determined by ESR were consistent with those obtained from CD, confirming the peptide's stronger interaction with sterol-containing membranes. Zeta-potential analysis showed reduced negative surface charge upon peptide addition, especially in anionic vesicles, supporting electrostatic interactions. A similar trend in PC/sterol vs. PG vesicles highlights the role of membrane fluidity and charge in Ctx(Ile²¹)-Ha peptide binding.