Extending the Hydrophobic Mismatch Concept to Amphiphilic Membranolytic Peptides

A series of nine amphiphilic, pore-forming α-helical KIA peptides (KIAGKIA repeats) with lengths between 14 and 28 residues were studied by solid-state ¹⁵N NMR to determine their alignment in oriented lipid bilayers. In a 2:1 mixture of 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidylcholine (DMPC) with its corresponding 1-myristoyl-2-hydroxy-<i>sn</i>-glycero-3-phosphocholine (lyso-MPC), which has a highly positive spontaneous curvature, the helix tilt angle was found to vary steadily with peptide length. The shortest peptide was aligned transmembrane and upright, while the longer ones successively became tilted away from the membrane normal. This behavior is in agreement with the hydrophobic matching concept, conceived so far only for hydrophobic helices. In 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphatidylcholine, with a negative spontaneous curvature, all KIA peptides remained flat on the bilayer surface, while the cylindrical DMPC lipids permitted a slight tilt. Peptide insertion thus depends critically on the intrinsic lipid curvature, and helix orientation is then fine-tuned by membrane thickness. A refined toroidal pore model is proposed

Influence of the Length and Charge on the Activity of α‑Helical Amphipathic Antimicrobial Peptides

Hydrophobic mismatch is important for pore-forming amphipathic antimicrobial peptides, as demonstrated recently [Grau-Campistany, A., et al. (2015) <i>Sci. Rep.</i> <i>5</i>, 9388]. A series of different length peptides have been generated with the heptameric repeat sequence KIAGKIA, called KIA peptides, and it was found that only those helices sufficiently long to span the hydrophobic thickness of the membrane could induce leakage in lipid vesicles; there was also a clear length dependence of the antimicrobial and hemolytic activities. For the original KIA sequences, the cationic charge increased with peptide length. The goal of this work is to examine whether the charge also has an effect on activity; hence, we constructed two further series of peptides with a sequence similar to those of the KIA peptides, but with a constant charge of +7 for all lengths from 14 to 28 amino acids. For both of these new series, a clear length dependence similar to that of KIA peptides was observed, indicating that charge has only a minor influence. Both series also showed a distinct threshold length for peptides to be active, which correlates directly with the thickness of the membrane. Among the longer peptides, the new series showed activities only slightly lower than those of the original KIA peptides of the same length that had a higher charge. Shorter peptides, in which Gly was replaced with Lys, showed activities similar to those of KIA peptides of the same length, but peptides in which Ile was replaced with Lys lost their helicity and were less active

Structural characteristics of TP10.

<p>Summary of features related to the bipartite character of the hybrid peptide TP10 (positions labeled with CF₃-Bpg are marked in red).</p

Membrane-bound structure of TP10, as derived by solid-state ¹⁹F

<p>-<b>NMR</b>. (A) The N-terminal region is intrinsically unstructured (green) and connected to the C-terminal α-helix (red). The amphiphilic helix is embedded in the lipid membrane with a tilt angle of τ≈55° and an azimuthal rotation angle of ρ≈120°. (B) The helical wheel projection of the C-terminal mastoparan part illustrates how the charged Lys residues (grey) point towards the aqueous phase, while the hydrophobic residues (yellow) face the interior of the membrane. The yellow box represents the bilayer (not drawn to scale, and without implying any particular insertion depth of the peptide within the bilayer).</p

Solid-state NMR spectra of TP10:

<p>(A) ¹⁹F-NMR spectra of TP10 labeled with <b><i>L</i></b><b>-</b>CF₃-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/L = 1∶50, 1∶200, and 1∶400) in oriented DMPC/DMPG (3∶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B₀ (indicated by an arrow). (B) Solid-state ³¹P-NMR spectra of the same samples as in (A), recorded before and after the corresponding ¹⁹F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state ¹⁹F-NMR spectra of the nine <b><i>L</i></b><b>-</b>CF₃-Bpg labeled TP10 analogs at P/L = 1∶400, from which the dipolar couplings of the CF₃-groups were obtained for the structure calculation. All experiments were performed at 40°C.</p

Fibril formation of TP10.

<p>TEM images of TP10 analogs (A) Leu16→ <b><i>L</i></b><b>-</b>CF₃<i>-</i>Bpg, (B) Leu16→ <b><i>D</i></b><b>-</b>CF₃-Bpg, showing a network of amyloid-like fibrils.</p

CD spectra of the CF₃-Bpg labeled TP10 analogs.

<p>CD spectra are recorded in the presence of unilamellar DMPC/DMPG (3∶1) vesicles at a P/L ratio of 1∶200. (A) <b><i>L</i></b><b>-</b>epimers and (B) <b><i>D</i></b><b>-</b>epimers are compared with the WT peptide (black line). Analogs with CF₃-Bpg in the galanin part are represented by green lines and in the mastoparan part by red lines.</p

OCD spectra of TP10.

<p>Representative OCD spectra of TP10 labeled with <b><i>D</i></b><b>-</b>CF₃-Bpg in oriented DMPC/DMPG (3∶1) bilayers at P/L = 1∶50, measured after 1, 5, and 8 days of ageing. (A) Peptide analogs with a substitution in the N-terminal region (here: position Leu4) have a predominantly α-helical structure, just like the WT peptide. (B) When <b><i>D</i></b><b>-</b>CF₃-Bpg is placed into the C-terminal region (here: position Leu16), the peptide aggregates with a β-sheet conformation typical of amyloid-like fibrils.</p

Cellular uptake of TP10.

<p>(A, B) Internalization of TP10 WT and of two representative ¹⁹F-labeled analogs Ile8→ <b><i>L</i></b><b>-</b>CF₃-Bpg (C), and Ile20→ <b><i>L</i></b><b>-</b>CF₃-Bpg (D) by HeLa cells. The cells were incubated with 10 µM peptide at 37°C for 30 min.</p

Secondary structure of TP10-WT bound to DMPC/DMPG vesicles evaluated from the CD spectrum (P/L = 1∶50, see Figure S1 A/B in File S1 for details).

a<p>NRMSD = normalized root mean square deviation between calculated and experimental CD spectra.</p