Label-free measurement of antimicrobial peptide interactions with lipid vesicles and nanodiscs using microscale thermophoresis

Antimicrobial peptides (AMPs) are a promising source of inspiration for new antibiotics discovery, in part because they believed to not trigger rapid resistance development since AMPs have co-existed with bacteria throughout evolution and do not target a single enzyme encoded by a single gene. AMPs is a diverse class of molecules that have diverse and often unspecific modes of action interfering with the membrane potential or the bacterial cell wall integrity, but also intracellular targets have been reported. Regardless of the mode of action(s), the AMPs will first have to interact with-, or penetrate through-, the bacterial cell well, and early characterization of AMP activity relies on the determination of the KD (dissociation constant for binding affinity) and the KP (partitioning constant) of AMP:lipid interactions. Here we demonstrate that microscale thermophoresis (MST) can be used for reliable unlabelled measurement of KD and KP utilising the intrinsic tryptophan fluorescence, thus removing the need for chromophore labelling. The MST results of binding to small unilamellar vesicles (SUVs) and stryrene maleic acid (SMA) based nanodiscs are compared to the corresponding surface plasmon resonance (SPR) measurements. SMA-QA nanodiscs are shown to be best suited for accurate measurements, while vesicles are a viable alternative. Unmodified SMA-nanodiscs proved unsuitable due to interactions between the cationic AMPs and the anionic polymer belt. Significant reduction of KD was observed when 5% anionic lipids were included in the lipid composition of the membrane models. This highlights the preference of the tested AMPs for anionic bacterial membranes, and the measured KD and KP values correlate well with their activity towards S. aureus and E. coli. We conclude that MST is a promising method for fast and efficient detection of peptide-lipid interactions, and the relative strength of the interactions can be reliably ranked within a library of screened compounds.