Studies of kinetics and equilibrium membrane binding of class A and class L model amphipathic peptides

Abstract

We studied the kinetics and equilibrium membrane binding of two amphipathic α-helical peptides: the 18L peptide, which belongs to the class L (lytic peptides), and the Ac-18A-NH 2 peptide of the class A (apolipoprotein), according to classification of Segrest et al. ((1990) Proteins, 8, 103–117). Both for cationic 18L and zwitterionic Ac-18A-NH 2, the presence of acidic lipids increased the membrane binding constants by two orders of magnitude. The free energy of peptide–membrane association was in the range of 8.5–12.8 kcal/mol. Binding isotherms corresponded to monomer partitioning with saturation at high peptide/lipid ratios. This was also supported by stopped flow studies of the kinetics of peptide-membrane association as measured by peptide tryptophan fluorescence or by energy transfer from the peptide to the lipid-anchored anthrylvinyl fluorophor. The apparent time required for peptide–membrane equilibration was in the millisecond range. At low peptide/lipid ratios it depended on lipid concentration and was independent of the peptide concentration. The rate of peptide–membrane association was found to be relatively close to the diffusion limit. Thus peptide–membrane affinity was mostly determined by the peptide dissociation rate, i.e. higher membrane affinity correlated with a decrease in dissociation rate and with a slower peptide exchange. We have shown that the dynamic character of the peptide membrane equilibrium can be used for selective peptide targeting and disruption of membranes with a specific lipid composition.