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.
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis
Cited by
16 articles.
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