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

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.

Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields

“…This occurrence may be caused by the shielding effect of NaCl between the cationic peptide and the zwitterionic membrane. Another possible reason is that Na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (Sabapathy et al, 2020). The reduced activity of synthetic peptide [RLLR] 5 under high salt concentration is possibly caused by the destruction of its α-helix structure.…”

Intracellular Ionic Strength Sensing Using NanoLuc

“…Similarly, the higher order assembly of the purified Caenorhabditis elegans LAF-1 protein formed by phase separation could be altered by changes in the concentration of sodium chloride in the solution [ 16 ]. Ionic strength also regulates the gene expression of osmolarity-sensitive genes, as detected by electron microprobe analysis and Northern blot assays of mRNA derived from various tonicity-sensitive genes [ 17 , 18 ], cellular volume by modulating the activity of ion channels [ 19 ], peptide–lipid bilayer interactions [ 20 ], and the diffusion of biomolecules on lipid membranes [ 21 ].…”

Efficacy of a Combined Antiplatelet Therapy Is Not Affected by a Simultaneous Binding of Cangrelor and PSB 0777 to Albumin

“…It could be noticed that cangrelor was used at 10-fold lower concentration than PSB 0777, which resulted from a difference in their IC 50 values required for effective inhibition of platelet function in whole blood: the apparent cell response to antagonist was achieved at nanomolar concentration of cangrelor and a micromolar concentration of PSB 0777 ( Boncler et al, 2018 ). The difference in concentration ranges, however, should not influence the results, because, in principal, the values of the rate constants and the equilibrium constants calculated from the rate constants are independent of the concentrations of both analyte and ligand ( Sabapathy et al, 2020 ).…”