Effects of Molecular Crowding on Membrane Binding of Buforin II and DesHDAP1

Antibiotic resistance is a serious global health concern, posing challenges to the effectiveness of traditional antibiotics in treating bacterial infections. To address this issue, researchers are exploring alternative therapeutic options, such as antimicrobial peptides (AMPs), which are natural defense molecules that exhibit a broad-spectrum of antimicrobial activity resulting in lower bacterial resistance. Buforin II (BF2), the most studied histone-derived antimicrobial peptide (HDAP), and DesHDAP1, an HDAP designed in our lab, share a mechanism of action. Both peptides cross membranes without causing cell lysis and appear to cause cell death through interactions with nucleic acids. However, most experiments measuring activity or mechanism of action of these peptides have been performed in dilute aqueous buffers that do not model the densely packed cellular environment. In previous studies, molecular dynamics simulations found that crowding agents hindered membrane interactions of DesHDAP1. In this study, we measured the binding affinity of DesHDAP1 and BF2 with large unilamellar vesicles (LUVs) in the presence and absence of crowders. Results indicate that crowders decrease membrane binding for both DesHDAP1 and BF2 experimentally. The difference in membrane binding between the environments with and without crowders emphasizes the importance of considering effects of extracellular crowding in research considering the behavior and mechanisms of antimicrobial peptides and in the efforts to design peptides with desirable antimicrobial and cell-penetrating properties.