Charge Distribution Fine-Tunes the Translocation of α-Helical Amphipathic Peptides across Membranes

Abstract

Hundreds of cationic antimicrobial and cell-penetrating peptides (CPPs) form amphipathic α-helices when bound to lipid membranes. Here, we test two hypotheses for the differences in the ability of these peptides to translocate across membranes. The first, which we now call the hydrophobicity hypothesis, is that peptide translocation is determined by the Gibbs energy of insertion into the bilayer from the membrane interface. The second, which we call the charge-distribution hypothesis, is that translocation is determined by whether the distribution of cationic residues in the peptide can transiently stabilize a high-energy inserted intermediate by forming salt bridges to the phosphates of lipid headgroups. To test these hypotheses, we measured translocation of two series of peptide variants. The first series was based on TP10W, a peptide derived from the amphipathic CPP transportan 10; the second was based on DL1a, a synthetic peptide derived from staphylococcal δ-lysin. The peptides in those two series had small sequence changes relative to TP10W and DL1a: either single-residue substitutions or two-residue switches, which were designed to increase or decrease translocation differently according to the two hypotheses. We found that with regard to the changes introduced in the sequences, five out of six peptide variants translocated in agreement with the charge-distribution hypothesis, whereas none showed agreement with the hydrophobicity hypothesis. We conclude that large effects on translocation are probably determined by hydrophobicity, but the fine tuning appears to arise from the distribution of cationic residues along the peptide sequence.

Figure 1

(A) The hydrophobicity hypothesis. Shown is the thermodynamic cycle for the interactions of a membrane-active peptide with a membrane. The peptide is shown in water (left) and on the membrane interface (bottom right) or inserted into the bilayer (top right)—no orientation is implied. The Gibbs energies in each step are $\Delta G_{\text{bind}}$, binding to the bilayer interface; $\Delta G_{\text{oct}}$, insertion into the bilayer core, approximated by transfer to octanol; $\Delta G_{\text{ins}}=\Delta G_{\text{oct}}-\Delta G_{\text{bind}}$, insertion into the bilayer interior; and $\Delta G_{\text{f}}$, folding in water. (B) The charge-distribution hypothesis. Shown is the final state of an MD simulation of TP10W placed in a lipid bilayer. The salt bridges between the lysine residues of the peptide and the phosphate headgroups of the lipids stabilize the high-energy inserted intermediate. The scheme in (A) was reprinted with permission from Yandek et al. (21). The image in (B) was reprinted with permission from Dunkin et al. (31). To see this figure in color, go online.

Figure 2

Concept of the experiment. GUVs prepared with inner vesicles are added to a solution containing peptide and a water-soluble fluorophore (green). The membrane of the vesicles is shown in red. Initially (A), the interior of the vesicles has no fluorescence (black circles). (B) The peptide induces flux into the outer vesicle. If the peptide does not translocate across the outer membrane, the inner vesicles remain dark (C). The appearance of fluorescence inside inner vesicles indicates that the peptide did translocate across the membrane of the outer vesicle (D). The scheme here was reprinted with permission from Wheaten et al. (26). To see this figure in color, go online.

Figure 3

Location of mutations in TP10W (left) and DL1a (right) variants. The structures were generated with the program PEPFOLD (63, 64, 65) and rendered with open-source PyMol. They do not represent the structures of the peptides on the membrane, which are mostly, but not fully, helical. To see this figure in color, go online.

Figure 4

Examples of dye flux into POPC GUVs containing inner vesicles. (A–C) Time series showing influx into GUVs added to a solution containing CF and a peptide. The pictures were taken at different times after addition of the GUVs to the peptide solution. (A) Ac-DL1a at 4.5, 13.5, 17.5, and 64.5 min. (B) Ac-DL1a-K26L at 2.5, 12.5, 40, and 77.5 min. (C) Ac-DL1a-G4-K10 at 5, 10.5, 33.5, and 39 min. The outer GUVs (leftmost images) have diameters of $\sim 50$–100 μm. The inner vesicles (rightmost images) have diameters of $\sim 10$–20 μm. (D–F) Plots show the dye flux into GUVs as a function of time for the same peptides in each series: (D) Ac-DL1a, (E) Ac-DL1a-K26L, and (F) Ac-DL1a-G4-K10. The red lines indicate flux into the outer GUV, and the black lines indicate flux into inner vesicles. To see this figure in color, go online.

Figure 5

(A–C) Binding kinetics for (A) Ac-TP10W, (B) Rh-TP10W, and (C) Ac-DL1a-G4-K10. In (A) and (C), FRET from Trp in the peptide to a lipid fluorophore (2 mol % 7MC-POPE) in POPC vesicles (1 μM peptide and 100 μM POPC LUVs). (B) Fluorescence intensity of the Rh-labeled peptide (26). (D) Dissociation kinetics of Ac-TP10W from POPC LUVs. The figure shows the decay in FRET from the Trp residue in the peptide to a lipid fluorophore present in the (donor) vesicles (0.5 μM peptide prebound to donor vesicles containing 2 mol % 7MC-POPE, mixed with 150 μM POPC LUVs containing no fluorophore). The data are shown in red. The black lines in (A), (B), and (D) are fits to single-exponential functions. In (C), the solid black line is a fit to a double exponential and the dashed line is a fit to a single exponential. To see this figure in color, go online.

Figure 6

Kinetics of peptide binding to lipid vesicles (POPC LUVs). The apparent rate constant, $k_{\text{app}}$, for the kinetic binding experiments is plotted as a function of lipid concentration to extract the on- and off-rate constants ($k_{\text{on}}$ and $k_{\text{off}}$) from the slope and the y-intercept of the line, respectively. The binding kinetics were measured using the change in FRET signal from an intrinsic Trp in the peptide to a lipid fluorophore (7MC-POPE) incorporated in the membrane. The data for the Rh-peptides are from (26). An experiment on one vesicle preparation is shown for each peptide, except in the case of Ac-DL1a-G4-K10, where the data from three independent experiments were combined. The error bars are standard deviations of $\sim 5$ curves for each data point (when not visible, they are contained inside the points). To see this figure in color, go online.

Figure 7

(A–J) CD spectra of the peptides (20 μM) bound to POPC vesicles (5 mM POPC LUVs). The spectra shown are averages of three independent experiments. Rh-TP10W and Rh-DL1a are included for comparison (26).