On the Origin of Multi-Exponential Kinetics in Peptide Binding to Phospholipid Vesicles

Abstract

Binding kinetics of amphipathic peptides to phospholipid bilayer vesicles are generally well described by a single exponential function. However, closer inspection of the data sometimes reveals the presence of additional exponential phases. These usually depend on the exact experimental conditions and on the peptide studied, but are indicative of other processes that contribute to the binding kinetics. We investigated the binding of a model amphipathic peptide, Lysette-26, to unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a function of lipid and peptide concentration. Lysette-26 is a 22 residue peptide with the sequence IISTIGDLVKWIIDTVKWIIDTVNKFTKK. The peptide binds well to zwitterionic lipid bilayer membranes and forms an amphipathic α-helix when bound at the membrane-water interface. Peptide binding was measured through fluorescence energy transfer from the intrinsic tryptophan residue in the peptide to an acceptor fluorophore embedded in the membrane at low concentrations. A series of exact kinetic models was developed to describe the experimental data. We found that the kinetics of peptide binding to POPC vesicles depended strongly on peptide concentration in solution. The model that best described the experimental data included the presence of peptide monomers and aggregates in solution that bind independently and with different rate constants to the lipid vesicles. Peptide aggregation is favored at higher peptide concentrations, leading to the appearance of an additional exponential phase. Based on the kinetic models, other processes, such as peptide insertion or translocation across the bilayer, could be excluded as the source of the second exponential phase in the binding kinetics.