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

Abstract

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (KD) and the partitioning coefficient between the aqueous- and membrane phases (KP) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of KD by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured KD values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs’ minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions or mapping of sample conditions in preparation for more advanced studies that rely on expensive sample preparation, labelling and/or instrument time.

Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance

“…The set was selected as a useful chemical model to study the effects of charge distribution on the interactions. The peptide c-LWwNKr ( Figure 5 ) was included in the study as a control that is chemically similar, but without antimicrobial activity (MIC > 256 μg/mL) ( Junkes et al, 2008 ; Bagheri, 2010 ; Gopal et al, 2012 ; Finger et al, 2015 ; Rainsford et al, 2022 ; Jakubec et al, 2023 ).…”

“…aeruginosa K34-7 strains were provided by The Norwegian National Advisory Unit on Detection of Antimicrobial Resistance (K-res), University Hospital of Northern-Norway (UNN). The peptides c-WKWKWK, c-WRWRWR, c-WWWKKK, c-WWWRRR and c-LWwNKr were synthesized as previously reported in ( Rainsford et al, 2022 ; Jakubec et al, 2023 ). PhosSTOP tablets were purchased from Roche via Sigma Aldrich.…”

“…For this sample optimal dilution was empirically determined by monitoring coverage of the chip. The SPR experiments were performed as was described in ( Rainsford et al, 2022 ; Jakubec et al, 2023 ). Briefly, the analysis was carried out with a T200 Biacore instrument (GE Healthcare, Chicago, Ill, United States), at 25°C.…”

“…Antimicrobial peptides (AMPs) are receiving increasing attention in the fight against AMR, as their different mode of actions lead to destabilization of bacterial membranes and bacterial death ( Czaplewski et al, 2016 ; Mahlapuu et al, 2016 ; Ageitos et al, 2017 ; Kumar et al, 2018 ). A test panel of five cyclic AMPs with various distributions of charged and hydrophobic amino acids, known to interact with bacterial membranes was used ( Junkes et al, 2008 ; Bagheri, 2010 ; Gopal et al, 2012 ; Finger et al, 2015 ; Rainsford et al, 2022 ; Jakubec et al, 2023 ). This set of peptides was originally designed to study how the peptide charge distribution affects membrane interactions and was used by our lab to explore kinetics of binding towards synthetic liposomes, nanodiscs and to a series of E .…”

“…This set of peptides was originally designed to study how the peptide charge distribution affects membrane interactions and was used by our lab to explore kinetics of binding towards synthetic liposomes, nanodiscs and to a series of E . coli strains ( Rainsford et al, 2022 ; Jakubec et al, 2023 ).…”