Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers

ConspectusThe structures of membrane-bound polypeptides are intimately related to their functions and may change dramatically with the lipid environment. Circular dichroism (CD) is a rapid analytical method that requires relatively low amounts of material and no labeling. Conventional CD is routinely used to monitor the secondary structure of peptides and proteins <i>in solution</i>, for example, in the presence of ligands and other binding partners. In the case of membrane-active peptides and transmembrane proteins, these measurements can be applied to, and remain limited to, samples containing detergent micelles or small sonicated lipid vesicles. Such traditional CD analysis reveals only secondary structures. With the help of an oriented circular dichroism (OCD) setup, however, based on the preparation of <i>macroscopically oriented lipid bilayers</i>, it is possible to address the membrane alignment of a peptide in addition to its conformation. This approach has been mostly used for α-helical peptides so far, but other structural elements are conceivable as well. OCD analysis relies on Moffitt’s theory, which predicts that the electronic transition dipole moments of the backbone amide bonds in helical polypeptides are polarized either parallel or perpendicular to the helix axis. The interaction of the electric field vector of the circularly polarized light with these transitions results in an OCD spectrum of a membrane-bound α-helical peptide, which exhibits a characteristic line shape and reflects the angle between the helix axis and the bilayer normal. For parallel alignment of a peptide helix with respect to the membrane surface (S-state), the corresponding “fingerprint” CD band around 208 nm will exhibit maximum negative amplitude. If the helix changes its alignment via an obliquely tilted (T-state) to a fully inserted transmembrane orientation (I-state), the ellipticity at 208 nm decreases and the value approaches zero due to the decreased interactions between the field and the transition dipole.Compared to conventional CD, OCD data are not only collected in the biologically relevant environment of a highly hydrated planar lipid bilayer (whose composition can be varied at will), but in addition it provides information about the tilt angle of the polypeptide in the membrane. It is the method of choice for screening numerous different conditions, such as peptide concentration, lipid composition, membrane additives, pH, temperature, and sample hydration. All these factors have been found to affect the peptide alignment in membrane, while having little or no influence on conformation. In many cases, the observed realignment could be related to biological action, such as pore formation by antimicrobial and cell-penetrating peptides, or to binding events of transmembrane segments of integral membrane proteins. Likewise, any lipid-induced conversion from α-helix to β-sheeted conformation is readily picked up by OCD and has been interpreted in terms of protein instability or amyloid-formation

¹⁹F‑Labeling of Peptides Revealing Long-Range NMR Distances in Fluid Membranes

NMR distance measurements lie at the heart of structural biology. However, long-range distances could not yet be detected in liquid–crystalline biomembranes, because dipolar couplings are partially averaged by the intrinsic molecular mobility. Using conformationally constrained ¹⁹F-labeled amino acids as reporter groups, we could more than double the accessible interatomic distance range by combining a highly sensitive solid-state multipulse ¹⁹F-NMR scheme with a favorable sample geometry. Two rigid 4F-phenylglycine labels were placed into the helical antimicrobial peptide PGLa embedded in fluid oriented membrane samples. A modified Carr–Purcell–Meiboom–Gill sequence yielded an intramolecular distance of 6.6 Å for the labels spanning one helix turn, and 11.0 Å was obtained when the labels spanned two turns. This approach should now also allow the characterization of conformational changes in membrane-active peptides and of oligomeric assemblies in a biologically relevant lipid environment

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

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

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

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

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

<i>D</i>-amino acid “scan” to identify aggregation-prone regions in TP10.

<p>Aggregation of TP10 depends on the position of substitution with the sterically restrictive <b><i>D</i></b><b>-</b>CF₃-Bpg, as monitored by solid-state ¹⁹F-NMR and OCD in oriented DMPC/DMPG (3∶1) at P/L = 1∶50. The boxed spectral regions show the static powder pattern contributions of immobilized molecules with −8 kHz splittings.</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