Abstract
Antimicrobial pore-forming peptides have the ability to selectively disrupt bacterial membranes which makes them promising agents against antibiotic-resistant pathogens. Here we probed their properties within various lipid membranes to decipher their sequence-function relationship, a critical step to optimize peptide selectivity and potency. Our main approach utilizes mass photometry and HDX-MS on peptides incubated with nanodiscs and liposomes of varying lipid composition mimicking either host-cell or pathogenic membranes.Mass photometry experiments comparing the mass of DPPC and DPPG nanodiscs revealed statistically significant mass differences between empty and peptide-incubated nanodiscs, indicative of peptide binding. HDX-MS showed reduced deuterium uptake when analyzing peptides in presence of membranes and revealed favored interactions between certain peptides and lipids. Examples include Melittin with E. coli polar extract and Maculatin 1.1 with POPC. The comparison of deuteration patterns of different peptides may also allow the determination of a peptide’s mode of action. Supporting methods such as CD spectroscopy were used to confirm membrane interactions by observing the transition from unfolded to a helical state upon membrane titration. Calcein-based leakage assays confirmed membrane disruption under the studied conditions.
Mass photometry and HDX-MS approaches are novel in their application to antimicrobial peptides, offer a framework to study peptide-lipid interactions and guide antimicrobial peptide design.