Mechanisms of antimicrobial, cytolytic, and cell-penetrating peptides: from kinetics to thermodynamics

Abstract

The mechanisms of six different antimicrobial, cytolytic, and cell-penetrating peptides, including some of their variants, are discussed and compared. The specificity of these polypeptides varies; however, they all form amphipathic alpha-helices when bound to membranes, and there are no striking differences in their sequences. We have examined the thermodynamics and kinetics of their interaction with phospholipid vesicles, namely, binding and peptide-induced dye efflux. The thermodynamics of binding calculated using the Wimley-White interfacial hydrophobicity scale are in good agreement with the values derived from experiment. The generally accepted view that binding affinity determines functional specificity is also supported by experiments in model membranes. We now propose the hypothesis that it is the thermodynamics of the insertion of the peptide into the membrane, from a surface-bound state, that determine the mechanism.

FIGURE 1

Models proposed for the mechanisms of peptides that cause all-or-none release (top) and graded release (bottom). The peptides are mostly unstructured in aqueous solution, but, as they bind to the membrane, form an amphipathic α-helix, which is shown in cross-section as a cylinder, the darker half-circles representing the hydrophobic face and the lighter, the hydrophilic face. All-or-none mechanism (top) (35): A, peptide in solution; B, peptide bound to the membrane surface of dye-loaded vesicles; C, peptide associated with vesicles in the pore state, which causes all-or-none efflux; and D, peptide bound to an empty vesicle, from which it dissociates back into solution. Graded mechanism (bottom) (19, 20): Binding of peptides creates a mass imbalance across the lipid bilayer, which perturbs the membrane, enhancing the probability of a peptide transiently inserting into the hydrophobic core and crossing the bilayer. In the bilayer-inserted state, the peptide causes dye efflux from the vesicle. As peptide translocation proceeds, the mass imbalance across the bilayer is dissipated and efflux slows down, eventually stopping. Reproduced, with modifications, from Biophysical Journal (20, 35, 36), with permission. Copyright 2007, 2008, and 2009, respectively, Elsevier.

FIGURE 2

Fluorescence requenching (ANTS/DPX) assay for cecropin A in POPC/POPG LUVs, bottom (35), and tp10 in POPC/POPS LUVs, top (20). The fluorescence quenching factor inside the vesicles, Q_in, is plotted against the fraction of fluorophore (ANTS) released, f_out. All-or-none release yields a horizontal line, indicating that the fluorescence inside the intact vesicles is independent of the amount of fluorophore released. Graded release yields a rising curve, because of relief of fluorescence quenching as DPX also leaks out of the vesicles. Reproduced, with modifications, from Biophysical Journal (20, 35), with permission. Copyright 2007and 2008, respectively, Elsevier.

FIGURE 3

Thermodynamic cycle for peptide binding to the membrane interface and inserting into the bilayer core. In water, the equilibrium between unfolded and folded conformations, governed by ΔG_f, is usually shifted to the unfolded state. Binding to the membrane interface is governed by ΔG_if (lower solid arrows). This is composed of two terms (dashed arrows), binding as an unfolded peptide and folding to a helix on the surface. Insertion into the bilayer core (ΔG_ins) can be approximated by ΔG_oct—if if ΔG_f is small. Reproduced, with modifications, from Biophysical Journal (20), with permission. Copyright 2007 Elsevier.

FIGURE 4

Reaction free energy diagram for the interaction of an amphipathic α-helical peptide with a phospholipid membrane. The meaning of the states is the same as in Figure 3.