Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers

Abstract

The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.

FIGURE 1

Helical wheel projection of Tp10 generated using the program Membrane Protein Explorer (44). Black symbols represent positively charged residues, white, hydrophobic residues, dark gray, hydrophilic (uncharged), and light gray, residues with intermediate polarity. The C-terminus is amidated; therefore it carries no charge.

FIGURE 2

Scheme of the model for peptide-induced transient pore formation and peptide translocation across the lipid bilayer. The α-helices are shown as cylinders (in cross section on the right), where the darker half-circles represent the hydrophobic faces and the lighter half-circles represent the hydrophilic faces. Also indicated are the apparent rate constants for each process. The mathematical translation of this scheme is given by Eqs. 2, 3, and 6–10. Binding of peptides creates a mass imbalance across the lipid bilayer, which perturbs the membrane, possibly because of increase in local curvature strain. This perturbation enhances the probability of a peptide transiently inserting into the bilayer hydrophobic core and eventually crossing the bilayer. In the bilayer-inserted state, the peptide catalyzes dye efflux from the vesicle lumen; this state constitutes the apparent ‘pore’. As peptide translocation is completed, the mass balance across the bilayer is restored and the rate of efflux becomes very slow or eventually stops.

FIGURE 3

Fluorescence of tp10-7mc as a function of its concentration in aqueous solution. The experiment was performed by serial dilution of the same solution. The inset shows the lower concentrations. Only perhaps at ∼40 μM is there a slight deviation from the linear dependence.

FIGURE 4

Binding kinetics of tp10-7mc to LUVs of POPC (A and C) and POPS/POPC 2:8 (B and D). The experimental decay of the fluorescence of tp10-7mc (7-methoxycoumarin chromophore) upon mixing with LUVs to a final lipid concentration of 80 μM is shown for POPC (A) and POPS/POPC 2:8 (B). The lines are single exponential fits (Eq. 4). Note that the timescale varies by a factor of 10 between the two types of vesicles. A plot of the apparent values of the rate constant in experiments such as those in (A) and (B), as a function of lipid concentration is shown for POPC (C) and POPS/POPC 2:8 (D). The values of the slopes correspond to k_on (Eq. 5), which are indicated in Table 1. Two samples were examined for POPC and three samples for POPS/POPC 2:8. For each sample, 3–5 curves were collected. The means and standard deviations from all values are shown. The major contribution to the variance arises from the different samples. When not shown, the standard deviation is inside the point, but most of these cases correspond to concentrations examined on the same sample.

FIGURE 5

ANTS/DPX assay to determine the mechanism of release. The quenching function inside, , which is the ratio of ANTS fluorescence inside the vesicle in the presence (F_i) and absence () of quencher (DPX), is plotted against the ANTS fraction outside the vesicles, f_out, corrected for incomplete entrapment as described in Ladokhin et al. (26). The horizontal line would be the result expected for an all-or-none release. Clearly, release is graded. The solid line represents a fit of Eq. 11 to the data points, which are pooled from three independent samples. The fit parameters are K_d = 50 M⁻¹, K_a = 180 M⁻¹, and α = 0.55.

FIGURE 6

(A) Kinetics of carboxyfluorescein efflux from POPC LUVs after tp10 (0.5 μM) addition. The gray curves, from top (fastest) to bottom (slowest) are the experimental data for 20, 30, 50, 100, and 200 μM lipid. The data were normalized by the Triton X-100 release levels. Each curve is the average of a total of 5–9 experiments performed on 2 or 3 independent samples. The thin black lines represent the best fit of the theoretical kinetic model to the experimental data, allowing for a 50% variation in each parameter relative to the best simultaneous fit. The values of the best global fit are indicated in Table 1. Because Triton X-100 release levels obtained with this peptide have shown a significant variation between different preparations, an amplitude factor of 0.75–1.2 was included in the fits. For the fits shown, the amplitude factors are 0.83, 0.97, 1.2, 1.2, and 1.2 for 20, 30, 50, 100, and 200 μM lipid, respectively. (B) Reverse experiment of carboxyfluorescein efflux from POPC LUVs induced by tp10. Donor vesicles were prepared by preincubating 1 μM tp10 with 60 μM POPC (empty LUVs) for 1 h, and the kinetics were followed by mixing with 40-μM POPC acceptor vesicles (containing dye) so that the final concentration of POPC was 50 μM and the final tp10 concentration was 0.5 μM in the stopped-flow experiment. The efflux fraction is normalized to the Triton X-100 release level. The amplitude factor was 0.92. The gray curve represents the experimental data, and the thin black line is the best (global) fit, with the same parameters as in (A), within the allowed variation (Table 1). Please note that the timescales are different in panels (A) and (B).

FIGURE 7

Same as for Fig. 6, but for vesicles of POPS/POPC 2:8. (A) Forward reaction. The peptide concentration is 0.5 μM and the lipid concentrations are 20, 30, 50, and 100 μM. The amplitude factors are 0.97, 0.98, 1.2, and 1.2, in the same order. (B) Reverse experiment. The amplitude factor is 0.75. Please note that the timescales are different in panels (A) and (B).

FIGURE 8

The fraction of the different peptide states as a function of time, calculated from the fits for 20 μM POPC (solid lines) and 20 μM POPS/POPC 2:8 (dashed lines). The tp10 concentration is 0.5 μM in both cases. (A) shows the fraction in aqueous solution; (B) shows the fraction of tp10 bound to the outer leaflet (decreasing curves) and to the inner leaflet (increasing curves) of the bilayer; and (C) shows the fraction of tp10 inserted into the bilayer. The latter curves should be viewed only qualitatively because of the high degree of correlation between k_dins and k_eflx, which can significantly alter the time and amplitude of the inserted fractions.

FIGURE 9

Thermodynamic cycle for the transfer of tp10 from water to the bilayer interior. The Gibbs free energies, given in kcal/mol on the figure, were calculated with the program Membrane Protein Explorer (44) (and verified by hand), based on the hydrophobicity scales of White and Wimley (23).