Revisiting the Interaction of Melittin with Phospholipid Bilayers: The Effects of Concentration and Ionic Strength.

Thiru Sabapathy,Evelyne Deplazes,Ricardo L Mancera

doi:10.3390/ijms21030746

Thiru Sabapathy, Evelyne Deplazes + Show 1 more

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https://doi.org/10.3390/ijms21030746

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Abstract

Melittin is an anti-microbial peptide (AMP) and one of the most studied membrane-disrupting peptides. There is, however, a lack of accurate measurements of the concentration-dependent kinetics and affinity of binding of melittin to phospholipid membranes. In this study, we used surface plasmon resonance spectroscopy to determine the concentration-dependent effect on the binding of melittin to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers in vesicles. Three concentration ranges were considered, and when combined, covered two orders of magnitudes (0.04 µM to 8 µM), corresponding to concentrations relevant to the membrane-disrupting and anti-microbial activities of melittin. Binding kinetics data were analysed using a 1:1 Langmuir-binding model and a two-state reaction model. Using in-depth quantitative analysis, we characterised the effect of peptide concentration, the addition of NaCl at physiological ionic strength and the choice of kinetic binding model on the reliability of the calculated kinetics and affinity of binding parameters. The apparent binding affinity of melittin for POPC bilayers was observed to decrease with increasing peptide/lipid (P/L) ratio, primarily due to the marked decrease in the association rate. At all concentration ranges, the two-state reaction model provided a better fit to the data and, thus, a more reliable estimate of binding affinity. Addition of NaCl significantly reduced the signal response during the association phase; however, no substantial effect on the binding affinity of melittin to the POPC bilayers was observed. These findings based on POPC bilayers could have important implications for our understanding of the mechanism of action of melittin on more complex model cell membranes of higher physiological relevance.

Highlights

Anti-microbial peptides (AMPs) are found throughout the animal and plant kingdom, where they form an important part of the innate immune system [1]
The POPC small unilamellar vesicles (SUVs) were deposited on the L1 sensor chip at a flow rate of 5 μL/min for 60 min, attaining an immobilization level up to a maximum of ~8400 RU
One of the aims of this study was to investigate the effect of melittin concentration on the apparent affinity of its interaction with POPC, the quality of fit to the data using different kinetic binding models and the subsequent reliability of the estimated binding constants

Summary

Introduction

Anti-microbial peptides (AMPs) are found throughout the animal and plant kingdom, where they form an important part of the innate immune system [1]. Like other membrane-disruptive peptides, AMPs are positively charged (cationic) and have a relatively large number of hydrophobic residues that are often clustered to give the peptide an overall amphipathic character [5,11,12,13,14,15] Their cationic nature enables electrostatic interactions with the negatively charged (anionic) lipid head groups found in bacterial membranes, while the amphipathic nature facilitates insertion into the hydrophobic core of the membrane. Characterising the membrane-binding properties of AMPs is important for the development of therapeutic peptides (e.g., antibiotics or anti-cancer agents) with higher specificity for microbes or cancer cells

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