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

11 November 2022, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Antimicrobial peptides (AMPs) are a promising source of inspiration for new antibiotic discovery, in part because they believed to not trigger rapid resistance. AMPs can target many features of the cell surface, and effective resistance development may require multiple mutations in parallel that have large fitness costs. Although they have diverse modes of action, AMPs will often begin by binding to the membrane; cell-penetrating peptides will subsequently need to cross it. Characterization of AMP activity can thus be inferred by the determination of KD (dissociation constant for binding affinity) and KP (partitioning constant). Here we demonstrate that microscale thermophoresis (MST) can be used for reliable label-free measurement of KD and KP utilising the intrinsic tryptophan fluorescence - removing the need for chromophore labelling. The MST results of binding to small unilamellar vesicles (SUVs) and styrene 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.

Keywords

Microscale Thermophoresis
Antimicrobial peptides
nanodiscs
Vesicles

Supplementary materials

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Supplementary Materials
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Supplementary materials related to peptide synthesis and experimental setup.
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