A membrane-translocating peptide penetrates into bilayers without significant bilayer perturbations

Abstract

Using a high throughput screen, we have identified a family of 12-residue long peptides that spontaneously translocate across membranes. These peptides function by a poorly understood mechanism that is very different from that of the well-known, highly cationic cell penetrating peptides such as the tat peptide from HIV. The newly discovered translocating peptides can carry polar cargoes across synthetic bilayers and across cellular membranes quickly and spontaneously without disrupting the membrane. Here we report on the biophysical characterization of a representative translocating peptide from the selected family, TP2, as well as a negative control peptide, ONEG, from the same library. We measured the binding of the two peptides to lipid bilayers, their secondary structure propensities, their dispositions in bilayers by neutron diffraction, and the response of the bilayer to the peptides. Compared to the negative control, TP2 has a greater propensity for membrane partitioning, although it still binds only weakly, and a higher propensity for secondary structure. Perhaps most revealing, TP2 has the ability to penetrate deep into the bilayer without causing significant bilayer perturbations, a property that may help explain its ability to translocate without bilayer permeabilization.

Figure 1

Amino-acid sequences of the translocating peptide TP2 and the inactive control ONEG. The amino acids that were deuterated for the neutron diffraction experiments are shown in bold and underlined.

Figure 2

CD spectra of 100 μM TP2 and ONEG in buffer and in the presence of 5 mM POPC liposomes. In both samples, TP2 has more secondary structure.

Figure 3

OCD spectra of 5 mol % TP2 and ONEG in oriented multibilayers of POPC, equilibrated at 76% relative humidity in a sealed chamber through the vapor phase.

Figure 4

Absolute neutron scattering length density profiles for bilayers with (top) 5 mol % TP₂ (solid line) and TP2-D (dashed line), as well as (bottom) 5 mol % ONEG (solid line) and ONEG-D (dashed line). The profiles are created using the absolute structure factors shown in Table 2.

Figure 5

Difference neutron scattering profiles, showing the transbilayer distribution of the deuterated amino acids in TP2 (top) and ONEG (bottom).

Figure 6

Leakage of the dye ANTS and the quencher DPX from vesicles in response to TP2 and ONEG. Lipid concentration is 1 mM total. Peptide concentrations are 2, 10, and 20 μM.

Figure 7

Normalized resistance and capacitance of polymer cushioned, surface-supported POPC bilayers after the addition of TP2. The peptide concentration was 100 μM, the lipid concentration was 60 μM, and the bound peptide/lipid was ∼1:200.