Designing a Novel Ultrashort Cyclic [R₃W₄V] Antimicrobial Peptide with Superior Antimicrobial Potential Based on the Transmembrane Structure to Facilitate Pore Formation

The clinical application of antimicrobial peptides (AMPs) is frequently hindered by the inherent limitations of linear peptides. Previous studies have primarily focused on the physicochemical properties of AMPs, and there is a scarcity of information regarding the transmembrane structure and interactions of AMPs with cell membranes and their antimicrobial activity. The present study is the first to propose that the backbone cyclization of linear R₃W₄V (l(R₃W₄V)) into the cyclic R₃W₄V (c[R₃W₄V]) form can enhance the stability of its transmembrane structure and consequently improve its antibacterial activity. The results of the bacterial inhibition assays performed herein demonstrated that the antibacterial activity of c[R₃W₄V] against <i>Staphylococcus aureus</i> and <i>Bacillus subtilis</i> was approximately 17-fold and 19-fold higher than that of l(R₃W₄V). The effect of c[R₃W₄V] on the structure of the bilayer membrane was further assessed via well-tempered bias-exchange metadynamics simulations and long-time conventional unbiased molecular dynamics simulations. This study demonstrated that the single c[R₃W₄V] peptide assumes a stable transmembrane configuration. Consequently, as the number of peptides accumulating in the membrane core increases at higher peptide-lipid ratios, a higher number of phospholipid headgroups embedded into the hydrophobic lipid core, leading to membrane fusion, permeabilization, and deformation of the upper and lower leaflets of the bilayer. The study provides a novel computational perspective on enhancing the antimicrobial efficacy of AMPs and highlights the importance of peptide-membrane structures, dynamics, and interactions in promoting the membrane-disruptive potential of peptides.