pH-Dependent Membrane Interactions of the Histidine-Rich Cell-Penetrating Peptide LAH4-L1

Abstract

The histidine-rich designer peptide LAH4-L1 exhibits antimicrobial and potent cell-penetrating activities for a wide variety of cargo including nucleic acids, polypeptides, adeno-associated viruses, and nanodots. The non-covalent complexes formed between the peptide and cargo enter the cell via an endosomal pathway where the pH changes from neutral to acidic. Here, we investigated the membrane interactions of the peptide with phospholipid bilayers and its membrane topology using static solid-state NMR spectroscopy. Oriented ¹⁵N solid-state NMR indicates that in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) 3:1 mol/mole and at neutral pH, the peptide adopts transmembrane topologies. Furthermore, ³¹P and ²H solid-state NMR spectra show that liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and POPC/POPS 3:1 liposomes retain a bilayer macroscopic phase even at the highest peptide concentrations investigated, with an oblate orientational distribution of the phospholipids at a peptide/lipid ratio of 1:5. At pH 5, as it occurs in the endosome, the alignment of LAH4-L1 at a peptide/lipid ratio of 1:25 is predominantly parallel to POPC/POPS 3:1 bilayers (prolate deformation) when at the same time it induces a considerable decrease of the deuterium order parameter of POPC/²H₃₁-POPS 3:1. In addition, when studied in mechanically supported lipid membranes, a pronounced disordering of the phospholipid alignment is observed. In the presence of even higher peptide concentrations, lipid spectra are observed that suggest the formation of magnetically oriented or isotropic bicelles. This membrane-disruptive effect is enhanced for gel phase DMPC membranes. By protonation of the four histidines in acidic environments, the overall charge and hydrophobic moment of LAH4-L1 considerably change, and much of the peptide is released from the cargo. Thus, the amphipathic peptide sequences become available to disrupt the endosomal membrane and to assure highly efficient release from this organelle.

Introduction

To quantitatively investigate the interaction contributions that determine the membrane topology of amphipathic helices, a family of histidine-rich peptides was designed using cationic antimicrobial peptides as templates (1). Many sequence variants of these LAH4 peptides have been prepared (2, 3), some members exhibiting potent antibiotic and antiplasmodial action (3), others have been shown to act as powerful nucleic transfection agents, with LAH4-L1 being the most efficient sequence for this activity (4). Thereby, LAH4 peptides constitute a family of cell-penetrating peptides that have been used to transfer not only pDNA, small interfering RNA (siRNA), and CpG oligonucleotides (5), but also quantum dots (6), adeno-associated viruses (AAVs (7)), lentiviruses used in ex vivo gene therapeutic approaches (2), and large proteins and peptides (8), where the latter have been used, e.g., in vaccination trials (5). LAH4 peptides associate with their cargo in a non-covalent manner (4, 9) and have been shown to transfer large biomacromolecules up to 500 kDa in size into cells in a fully functional state (8).

The core of the LAH4 peptides consists of alanines and leucines with four histidines interspersed in such a manner that in membrane environments, the peptides form amphipathic helices where all four histidines are localized on one face (the sequence of LAH4-L1 is shown in the Materials and Methods). Two lysines at each terminus assure good solubility of the peptides in aqueous environments and serve as membrane interfacial anchors or as nucleic acid contact sites (10). In membrane environments, the histidine side chains of the parent compound LAH4 exhibit pK values between 5.4 and 6.0, and thereby, these residues, by changing the charge and hydrophobic moment of these helices in a pH-dependent manner, have a pronounced influence on how the peptides arrange in lipid bilayers (11). As a consequence, LAH4 aligns parallel to the surface of POPC bilayers at pH <6 but adopts transmembrane orientations under neutral conditions when the histidines are uncharged (12). NMR structural investigations have revealed a flexible hinge region at intermediate pH, facilitating membrane insertion during the in-plane to transmembrane transition (11). The transition is reversible and has been used to quantify the transfer energy of amino acid side chains from the membrane interface to the membrane interior (13, 14). Since the development of LAH4 (1), an aspartate-rich peptide has been discovered that also exhibits pH-dependent membrane topology with potential biomedical applications (15), and a series of peptides designed to form pH-dependent macromolecular pores in membranes has been presented recently (16, 17).

Dynamic light scattering (DLS), circular dichroism spectroscopy, and fluorescence self-quenching indicate that in aqueous buffers, LAH4 and LAH4-L1 form small α-helical aggregates at neutral pH that dissociate into extended monomers at lower pH (18, 19). This pH-dependent transition between helical and random-coil conformation of a number of LAH4 derivatives has been studied in quantitative detail (20).

LAH4-L1 and related peptides exhibit excellent DNA (21) and siRNA transfection activities, with higher efficiencies than that of commonly used compounds such as lipofectamine, 1,2-dioleoyl-3-trimethylammonium-propane, and polyethylenimine (9). When the peptides and DNA are mixed at pH 7.4, transfection complexes are formed that enter the cells via an endosomal pathway (21). Notably, size measurements indicate that the dimensions of the complexes depend on the detailed experimental conditions and can be modulated, for example, by the salt concentration (22). Thus, a large range of hydrodynamic diameters of these overall positive complexes can be obtained, ranging from 100 nm to micrometers (22). Isothermal titration calorimetry indicates that in these complexes, one peptide is associated with approximately two basepairs of DNA (23). ¹³C magic-angle-spinning (MAS) solid-state NMR chemical shift measurements show that LAH4 also retains an α-helical conformation in the transfection complex (23). Furthermore, rotational-echo double-resonance distance measurements indicate that the positive lysine termini of the peptide interact with the negatively charged phosphates of the DNA, whereas the histidines are farther apart (10). Thereby, the two lysine side chains at each terminus interconnect distant sections of the extended DNA molecule, or different DNA strands, leading to the observed condensation of this macromolecule.

It has been shown that transfection of DNA can be inhibited by bafilomycin, a blocker of endosomal acidification, indicating that the peptide-nucleic acid complex enters via an endosomal pathway and that acidification of this organelle is an essential step (21). The association of LAH4 peptides with DNA, which occurs in the micromolar range, is driven by electrostatic interactions and is reversible (23). Importantly, at pH <6, in correspondence with conditions in the endosomal compartment, the histidine side chains become positively charged and about half of the peptides are released from the transfection complex (23). The liberated LAH4 peptides are now available within the endosomal compartment at high concentrations, where they can potentially interact with the membranes of this organelle. Notably, endosomal escape is an essential step during the transfection process and is often limiting for cargo delivery (24).

Although a membrane-lytic activity has been suggested to be key to explain the potent and widely applicable cell-penetrating activity of LAH4 peptides, this hypothesis has not been supported by experimental data that would confirm the effect of LAH4 or LAH4-L1 on membrane integrity. Therefore, to close this gap, in this work, the membrane interactions at the high peptide concentrations that occur in the endosome are analyzed at neutral and acidic pH. Here, using solid-state NMR spectroscopy, we investigated in quantitative detail the interactions of the peptides with membranes and how such interactions are potentially involved in the endosomal escape process. On one hand, ³¹P solid-state NMR is a well-established method to monitor the macroscopic phase properties of phospholipid membranes where the characteristic line shapes reveal the presence of bilayer, hexagonal, or isotropic phases (25, 26). More recently, the method has been refined to also monitor magnetic deformation or alignment of membrane components that occur, for example, in elongated vesicles or for bicellar structures (27, 28, 29). On the other hand, ²H solid-state NMR allows one to study the fatty acyl chain order parameters of deuterated lipids and thereby reveals interesting details on how peptides and other membrane-active components affect the membrane structure (26, 30, 31, 32). In combination with refined deuteration schemes, the method has also been used to identify selective interactions of membrane-active peptides with only one component within mixed membranes (4).

Finally, techniques involving solid-state NMR spectroscopy of polypeptides labeled with ¹⁵N and ²H and reconstituted into uniaxially oriented membranes have been developed to obtain detailed information on the structure, topology, and dynamics of membrane-active peptides (33, 34, 35). Notably, the ¹⁵N chemical shift provides a direct indicator of the approximate tilt angle of helical peptides, where values around 200 ppm show a transmembrane topology, whereas chemical shifts of <100 ppm are associated with helices oriented parallel to the membrane surface. All of these solid-state NMR approaches have been used here to reveal, in quantitative and comprehensive detail, how LAH4-L1 interacts with phospholipid membranes, resulting in macroscopic changes in membrane supramolecular assembly, thereby assuring the highly efficient escape of cargo from the endosomal compartment.

Materials and Methods

Phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC; T_m = 23°C), 1,2-dimyristoyl-d₅₄-sn-glycero-3-phosphocholine (²H₅₄-DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; T_m = −2°C), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS; T_m = 14°C), and 1-palmitoyl-d₃₁-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (²H₃₁-POPS) were from Avanti Polar Lipids (Alabaster, AL) and were used without further purification. Chloroform, methanol, and trifluoroethanol were from Sigma-Aldrich (Lyon, France).

Peptide synthesis

The peptide LAH4-L1 (KKALL AHALH LLALL ALHLA HALKK A-CONH₂; molecular mass 2778.48 Da) was prepared by standard Fmoc solid-phase peptide chemistry using a Millipore 9050 automatic peptide synthesizer (Eschborn, Germany). A batch was prepared where the ¹⁵N-labeled Fmoc-leucine analog was used at the coupling step of the Leu-14 position (underlined in sequence). After cleavage from the resin, the peptide was purified by reverse-phase high performance liquid chromatography with an acetonitrile/water gradient of increasing hydrophobicity (in the presence of 0.1% trifluoroacetic acid). To exchange the trifluoroacetic acid counterions, the main peak was collected, resolubilized in 4% acetic acid, and lyophilized (36). The identity and purity of the peptide were controlled by matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry.

Preparation of multilamellar vesicles

A mixture of POPC/²H₃₁-POPS 3:1 or DMPC/²H₅₄-DMPC 4:1 was prepared and solubilized in chloroform/methanol (2:1 v/v). Either ∼5 or 13 mg (depending on the capacity of the 3.2 or 4 mm MAS rotors, respectively, used as containers) of lipid per sample was mixed with different amounts of LAH4-L1 (previously solubilized in chloroform/methanol 2:1 v/v) at the desired molar peptide/lipid (P/L) ratios. The solvent was gently evaporated under a stream of nitrogen to obtain a lipidic film along the walls of the glass tube. The film was dispersed in MilliQ water and the pH was equilibrated at ∼5 or 7.4 by adding 0.5 or 0.1 M NaOH, respectively, and lyophilized. Because of the acidic condition during the purification of the peptide and the small sample volume, this initial adjustment assures an accurate equilibration of the pH of the final sample without the need to modify the volume and concentrations after addition of the buffer. Thereafter, the samples were solubilized in either 100 mM Tris buffer (pH 7.4) or in 100 mM acetate (pH 5) to reach a lipid concentration of 160 mM. Finally, the multilamellar vesicles (MLVs) were equilibrated by three freeze/thaw cycles (1 min in liquid nitrogen, 1 min at ambient temperature, and 10 min at 45°C, with vortexing).

It should be noted that in a previous study, a binding constant of 1.4 × 10⁶ M⁻¹ was determined where the peptide associates with four POPS lipids of POPC/POPS membranes, and 3 × 10⁴ M⁻¹ for POPC at pH 5.5 where the peptide associates with six lipids (37). Assuming similar values for the membranes studied here virtually all peptides should be membrane associated, except at the highest P/L ratios, where the binding sites are saturated.

Uniaxially oriented samples were prepared according to previously described protocols (36). About 80 mg of lipids were dissolved at a molar ratio of POPC/POPS 3:1 in chloroform/methanol 4:1 v/v. The corresponding amount of peptide was dissolved in 300–500 μL of trifluoroethanol and added to the lipid mixtures. The pH was adjusted by addition of microliter amounts of 100 mM NaOH and 1 mM EDTA. The solvent was partially evaporated and the mixture was dried onto 20–25 glass plates (8 × 21 mm) (Marienfeld, Lauda-Königshofen, Germany), first by exposure to air, then for at least 3 h under high vacuum to remove any traces of organic solvent. The glass plates were placed in a hydration chamber at 93% relative humidity at ambient temperature for at least 10 h. The pH of the samples was again tested from a small part of the sample that was mixed with 100 μL of deionized water. After equilibration, the glass plates were stacked on top of each other, wrapped with Teflon tape, and sealed in the polymer-barrier film Escal (Mitsubishi Gas Chemical, Tokyo, Japan).

Solid-state NMR spectroscopy

Proton-decoupled ³¹P Hahn-echo spectra (38) of MLVs were obtained on a Bruker Avance 500 NMR spectrometer equipped with a 4 or 3.2 mm MAS probe head (Bruker Biospin, Rheinstetten, Germany) without spinning at 37 and 7°C for DMPC samples, or 30°C for POPC/POPS samples. The following parameters were used: 90° pulse between 5 and 6.12 μs; echo delay, 100 μs; acquisition time, 40 ms; spectral width, 100 kHz; time-domain data points, 8192; number of scans, 96–1024 with the 4 mm probe or 160–4558 with the 3.2 mm probe; recycle delay, 10 s; and continuous wave ¹H decoupling at 28 or 60 kHz. Oriented samples were investigated on a Bruker Avance 400 spectrometer using a flat-coil probe (39). The ³¹P 90° pulses were 4 μs, the echo delay 40 μs, the recycle delay 2 s, and 32–64 scans were accumulated under spinal64 ¹H decoupling at 38 or 63 kHz. The spectra were processed with a line broadening of 100 Hz before Fourier transformation. 85% H₃PO₄ was used as an external reference (0 ppm).

For 2H solid-state NMR a solid quadrupolar echo-pulse sequence (40) was used with the following parameters: 90° pulse, 8 μs; echo delay, 40 μs; acquisition time, 6.5 ms; dwell time, 0.1 μs; a 250 kHz analog filter for noise reduction; time-domain data points, 65,536; number of scans, between 2750 and 190,000; and recycle delay, 0.3 s. The spectra were processed with a line broadening of 100 Hz before Fourier transformation.

The following equation was used to calculate the order parameter S_CD for each of the C-D bonds: $\Delta {\upsilon }_{\text{Q}}=\left(3/4\right)\left(e^{2}qQ/h\right)S_{\text{CD}}$, where $\left(e^{2}qQ/h\right)$ is the static quadrupole splitting constant, equal to 167 kHz for a C-D bond and $\Delta {\upsilon }_Q$ the observed quadrupolar splitting (30, 41, 42). The quadrupolar splitting of each C-D bond of the aliphatic chain of the labeled lipids was extracted using the TopSpin NMR software (SOLA (Solid Lineshape Analysis), version 2.2.4, Bruker Biospin). The QUAD all mode was used, where the full line shape of the spectrum of each CD₂ site was optimized by intensity and observed quadrupolar splitting to obtain the best representation of the experimental data.

The ¹⁵N solid-state NMR experiments of the peptide, labeled with ¹⁵N at a single site, were performed on a Bruker Avance 400 spectrometer, equipped with a static solid-state NMR probe with a flattened coil for mechanically oriented samples (39). Proton-decoupled ¹⁵N solid-state NMR spectra were acquired using a cross-polarization sequence with a contact time of 800 μs (43) and spinal64 ¹H decoupling. The B₁ fields were in the range 35–43 kHz, the recycle delay was 2 s, the spectral width was 32,000 Hz, the pre-acquisition delay was 20 μs, and the number of scans was 23,000–100,000. Before Fourier transformation, an exponential apodization function corresponding to a line broadening of 200 Hz was applied. ¹⁵NH₄Cl was used as a reference (39.3 ppm (44)).

Results

To investigate the macroscopic behavior of model lipid bilayers as a function of P/L ratio, proton-decoupled ³¹P solid-state NMR spectra were acquired for POPC/POPS 3:1(mole/mole) (Fig. 1) and DMPC phospholipid bilayers (Fig. 2). In the absence of peptide and at 30°C, the POPC/POPS 3:1 spectra exhibit the typical symmetric chemical-shift anisotropy of phospholipid membranes in their liquid crystalline state (26, 32), where the superimposition of two powder-pattern line shapes results in a shoulder at about −17 ppm and a less well-defined discontinuity around 30 ppm (Fig. 1, A and F). At pH 5, the addition of intermediate amounts of LAH4-L1 results in distorted powder-pattern line shapes (Fig. 1, G and H) representing a prolate distribution of phospholipid alignments relative to the magnetic field direction (45). When the P/L ratio is further increased to 1:12.5 (data not shown) or 1:5, broad isotropic peak intensities are observed at 30°C (Fig. 1 K). These are indicative of supramolecular assemblies such as micelles or isotropic bicelles that tumble fast enough to average the chemical shift anisotropy. Notably, an oriented spectral line shape is observed at −14.5 ppm when the temperature of the 1:12.5 P/L mixture is decreased to 16°C, where all phospholipids align with their long axis perpendicular to the magnetic field direction of the NMR spectrometer (Fig. 1 I). At pH 7.4, upon the addition of peptide (P/L ≤ 1:12.5), a symmetric powder-pattern line shape is observed, where both phospholipids exhibit closely related chemical shift anisotropies of 43.5 ± 1 ppm (Fig. 1, B–D). When the P/L ratio is increased to 1/5, the δ_|| intensities (at ∼29 ppm) are much more pronounced (Fig. 1 E), indicative of an oblate orientational distribution of the lipids, i.e., the fraction of lipids that is aligned parallel to the magnetic field direction is increased when compared to a statistical distribution.

Figure 1.

³¹P solid-state NMR spectra of POPC/POPS 3:1 membranes acquired at 11.8 Tesla in the absence (A and F) and presence of increasing amounts of LAH4-L1 at pH 7.4 (A–E) and pH 5 (F–K). The P/L ratios are indicated. The temperature was set to 30°C (303 K) except for the spectrum in (I), which was recorded at 16°C. At 30°C, this same sample exhibits an isotropic resonance (data not shown).

Figure 2.

³¹P solid-state NMR spectra of DMPC membranes acquired at 11.8 Tesla in the absence (A and H) and presence of increasing amounts of LAH4-L1 at pH 7.4 (A–G) and pH 5 (H–O). The P/L ratios are indicated. The temperature was set to 37°C (310 K; first and third columns) or 7°C (280 K; second and fourth columns).

In a second series of experiments, we also investigated the interactions of LAH4-L1 with DMPC membranes (Fig. 2). The phase-transition temperature of this lipid bilayer is 23°C, which allows one to compare the interactions of the liquid crystalline state and the gel phase more easily. In the liquid crystalline state (T = 37°C) and in the absence of peptide, the DMPC chemical shift anisotropy is ∼45 ppm (Fig. 2, A and H), in agreement with results from previous investigations (32). Whereas at pH 7.4 the liquid crystalline DMPC vesicles exhibit powder-pattern line shapes at all P/L ratios investigated, spectral features of oriented or isotropic bicelles are observed at P/L ≥ 1:12.5 (Fig. 2, M–O, lefthand plots), comparable to observations in POPC/POPS membranes (Fig. 1, I and K). Features of deformed/partially oriented liposomes are also observed in the absence of peptide or at low P/L ratio (Fig. 2, B and H), in agreement with previous publications (45), although the details of these line shapes are less well reproducible. Furthermore, at pH 7.4 and P/L 1:5 the δ_|| intensities are more accentuated (Fig. 2 G), suggesting an oblate deformation, albeit they are less pronounced for DMPC than for POPC/POPS bilayers (Fig. 1 E).

When the temperature is lowered to 7°C, i.e., well below the phase transition temperature, the chemical shift anisotropy increases to 68 ppm in the absence of peptide and at P/L ratios of 1:50 (Fig. 1, A, B, H, and I). However, at both pH values, a sharp isotropic line shape is apparent for all P/L ratios >1:50 suggestive of micellar or isotropic bicelle arrangements.

The ³¹P spectrum of MLVs in their liquid crystalline state exhibits a symmetric powder-pattern line shape (Figs. 1, A–D and F–H, and 2, A–L), where the chemical shift anisotropy is characterized by two motionally averaged components, δ_|| and δ_⊥ (∼30 and −15 ppm, respectively; Figs. 1 and 2), that derive from the three principle elements of the static chemical shift tensor, δ₁₁, δ₂₂, and δ₃₃ (46, 47). Because the intensity of the δ_|| shoulder is considerably reduced when the membranes are deformed or anisotropic bicelles orient in the magnetic field (Figs. 1, H and I and 2, B, H, M, and N), and therefore difficult to determine precisely, the δ⊥ value was extracted for each spectrum and is shown as a function of the P/L molar ratio in Fig. 3. The isotropic resonance position separates the powder-pattern line shape in such a manner that the δ_||/δ⊥ ratio of liquid crystalline phosphatidylcholines is −2 (32). Therefore, the alterations of δ⊥ also reflect changes in the chemical shift anisotropy. Upon addition of peptide, the δ⊥ position of the DMPC membrane increases continuously for both pH values, thereby approaching the isotropic resonance position. Clearly, at pH 5 the decrease of the latter is considerably more pronounced and occurs at lower P/L ratios. For POPC/POPS 3:1 (mole/mole), only the δ⊥ position of the maximal intensity could be determined, because the powder patterns and isotropic line positions of the two phospholipid components are superimposed. For both pH values, δ⊥ decreases slowly, until at pH 5 an isotropic resonance position is observed for P/L ratios of 1:12.5 and 1:5.

Figure 3.

The δ⊥ value of POPC/POPS 3:1 and DMPC membranes as a function of LAH4-L1/lipid molar ratio extracted from the ³¹P solid-state NMR spectra shown in Figs. 1 and 2. The temperature was set to 30°C (303 K) for POPC/POPS and to 37°C (310 K) for DMPC.

To better define the phospholipid supramolecular assemblies in the presence of LAH4-L1, the NMR DMPC samples were diluted 1000-fold and investigated by DLS at 25°C (Fig. S1). Whereas the DMPC vesicles exhibit a large hydrodynamic diameter around 1000 nm at both pH values, addition of LAH4 at ratios of 1:25 to 1:12.5 results in a 10-fold reduced size of the liposomes with highly polydisperse distribution. When more peptide is added, the size of the aggregates reaches the 10 nm range at a P/L ratio of 1:5.

To assess the effect of an increasing concentration of peptide at the level of the lipid fatty acyl chains, ²H solid-state NMR spectra were acquired from the same, unoriented samples carrying fully deuterated palmitoyl (²H₃₁-POPS; Fig. 4) or myristoyl (²H₅₄-DMPC; Fig. S2) chains. The spectra shown in Figs. 4 and S2 are composed of signals from different CD₂ and one CD₃ segment, each contributing a quadrupolar powder pattern, with the distance between the two main peaks defining a characteristic quadrupolar splitting for each site (30, 42). Without motions, the static splitting would amount to 125 kHz, but in a liquid crystalline bilayer, motions and cis-trans isomerization result in considerable averaging. This effect is also expressed by the deuterium order parameter, S_CD, representing the ratio between the measured and the maximal values. Notably, the segments exhibit more motional freedom in the hydrophobic interior when compared to the region of the carbonyls. Therefore, the order parameters tend to decrease for these segments.

Figure 4.

²H solid-state NMR spectra acquired at 11.8 Tesla for POPC/²H₃₁-POPS 3:1 in the absence (A and F) and presence of increasing amounts of LAH4-L1 at pH 7.4 (A–E) and pH 5 (F–K). The P/L ratios are indicated. The temperature was set to 30°C (303 K), except for (I), where it was set to 16°C (289 K).

The ²H solid-state NMR spectra of POPC/²H₃₁-POPS 3:1 (mole/mole) at pH 7.4 change in two complementary ways upon addition of peptide at ratios of 1:50 and 1:25 (Fig. 4, B and C). First, the individual resonances defining quadrupolar splittings broaden, and second, the quadrupolar splittings decrease. When more peptide is added, the effect is reversed, with a narrowing of the individual resonances and an increase in the quadrupolar splittings (Fig. 4, D and E). Furthermore, at a P/L ratio of 1:5, a second set of peaks is observed and the shoulders (reaching up to ±27 kHz) are accentuated. When the same sample is recorded at 60°C, individual peak intensities become distinguishable also within the shoulder (data not shown), confirming an oblate orientational distribution, as observed for the corresponding ³¹P spectrum (Fig. 1 E).

A gain in spectral resolution associated with line narrowing is also observed at pH 5 at P/L ratios of 1:50 and 1:25 (Fig. 4, G and H). At pH 5 and 30°C (303 K), at P/L ratios of 1:12.5 (data not shown) and 1:5, an isotropic peak is observed (Fig. 4 K). However, at 16°C, the ²H spectrum of the POPC/POPS membrane in the presence of a P/L ratio of 1:12.5 represents a prolate distribution of lipid alignments (Fig. 4 I). When the deuterium solid-state NMR spectra of ²H₅₄-DMPC are investigated (Fig. S2), they parallel the orientational distributions of the corresponding ³¹P solid-state NMR spectra (Fig. 2). The ²H₅₄-DMPC spectra (Fig. S2) also parallel many features described above for the POPC/²H₃₁-POPS 3:1 membranes (Fig. 4).

From the ²H solid-state NMR spectra shown in Figs. 4 and S2, the quadrupolar splittings were extracted and the order parameters S_CD plotted against each carbon position of the labeled lipids (Fig. 5). At pH 7.4, when LAH4-L1 is added to POPC/²H₃₁-POPS 3:1 membranes at P/L ratios of 1:50 or 1:25, a modest decrease of the order parameters is observed for all the C-²H₂ positions (Fig. 5 A). As expected, for the 1:50 sample, the shift is less pronounced than at the higher peptide concentration of 1:25. However, when the P/L ratio is further increased to 1:12.5 the order parameters are again close to the reference values except for carbon positions 4–6, which remain lower than the reference. At pH 5 (Fig. 5 B), the decrease in order parameters is more pronounced except for positions 2–3 at the ratio 1:12.5, where order parameters near the reference value are observed.

Figure 5.

The deuterium order parameter as a function of carbon position of POPC/²H₃₁-POPS 3:1 (A and B) and ²H₅₄-DMPC (C and D) in the presence of increasing amounts of LAH4-L1 at pH 7.4 (A and C) or at pH 5 (B and D). The temperature was set to 30°C (303 K) for POPC/POPS and 37°C (310 K) for DMPC. To see this figure in color, go online.

When compared to ²H₃₁-POPS embedded in a POPC membrane, the effects are less pronounced for ²H₅₄-DMPC. At pH 7.4, addition of peptide causes only small changes in the order-parameter profile (Fig. 5 C), with a small decrease for the first C-²H₂ positions and a small increase from carbon position 7 onward. At pH 5, a decrease in the order parameter is observed for the first carbon segments at P/L 1:25, and decreases throughout the fatty acyl chains only for the sample at a P/L ratio of 1:12.5 (Fig. 5 D).

To better understand the pH-dependent interactions of the peptide with the membranes, the LAH4-L1 sequence was prepared with a single ¹⁵N label at the Leu-14 position, reconstituted into uniaxially oriented lipid bilayers and investigated by ¹⁵N solid-state NMR spectroscopy. When the membrane normal is aligned parallel to the magnetic field direction of the NMR spectrometer, the ¹⁵N chemical shift is a direct indicator of the approximate orientation of helical domains relative to the lipid bilayer and provides valuable information about the peptide topology and thus the peptide-lipid interactions (33). Whereas chemical shifts around 200 ppm are indicative of transmembrane alignments, helices that are oriented along the bilayer surface exhibit resonances of <100 ppm. Fig. 6, A and B, shows the ¹⁵N chemical shift spectrum of LAH4-L1 obtained in oriented POPC/POPS 3:1 mol/mole bilayers at pH 7.4 at P/L ratios of 1:50 and 1:25, respectively. Notably, at these peptide concentrations, the MLVs remain undisrupted by LAH4-L1 at both pH values investigated (Fig. 1, B, C, G, and H). The ¹⁵N chemical shift is 196 ± 3 ppm and 202 ± 2 ppm, respectively (Fig. 6, A and B). When the same peptide is investigated at pH 5, the ¹⁵N chemical shifts are 88 ± 3 and 80 ± 1 ppm at P/L ratios of 1:50 and 1:25, respectively (Fig. 6, C and D). Theses data indicate that the peptide changes its topology in a pH-dependent manner from a transmembrane alignment at pH 7.4 to an orientation parallel to the surface when histidines are charged in an acidic environment. Notably, at pH 5, the resonance of the more concentrated samples is considerably sharper (line width at half height (LWHH), 12 ppm) when compared to the P/L 1:50 preparations (LWHH, 48 ppm) indicating that at the labeled site they possess a more uniform structure and topology. Similarly, the spectrum of Fig. 6 A shows contributions of ∼88 ppm, which can be assigned to a fraction of peptide adopting an in-planar orientation or a non-oriented powder-pattern line shape. A corresponding signal of the P/L 1:25 sample is considerably less intense (maximum at 80 ± 2 ppm).

Figure 6.

Proton-decoupled ¹⁵N (A–D) and corresponding ³¹P solid-state NMR spectra (E–H) of [¹⁵N-Leu14]-LAH4-L1 reconstituted in POPC/POPS 3:1 mol/mole bilayers oriented with the membrane normal parallel to the magnetic field direction. Spectra in the first and third columns are at pH 7.4 (A, B, E, and F) and those in the second and last column are at pH 5 (C, D, G, and H). The P/L ratio is 1:50 in the first row, (A, C, E, and G) and 1:25 in the second row (B, D, F, and H).

The corresponding ³¹P solid-state NMR spectra show two predominant peak intensities at 25–29 and 35–39 ppm, indicative of POPC and POPS phospholipids in their liquid crystalline state that align parallel to the sample normal. Judging from the intensities, the latter ³¹P chemical shift intensities are assigned to the POPS component, whereas phosphatidylcholine exhibits an anisotropy of 41 ppm in this mixture (cf. also Fig. 1; (26, 32)). Some intensities extend to −15 ppm (Fig. 6, E–H), demonstrating that the peptide causes other lipid alignments and/or conformational changes at the level of the lipid headgroup, i.e., the peptide exerts a disordering effect on the lipid bilayer (26). This effect is particularly pronounced for the P/L 1:25 sample at pH 5 (Fig. 6 H) despite the comparatively uniform orientation of the peptide (Fig. 6 D).

When the alignment of LAH4-L1 in POPC is investigated at a P/L ratio of 1:50, a single sharp intensity is observed at 76 ± 1 ppm (LWHH, 6.5 ppm) at pH 5, indicative of a homogenous in-planar orientation (Fig. S3 A) similar to the topology in POPC/POPS (Fig. 5 C). However, at pH 7.4, only 45% of the intensity is associated with a resonance around 175 ppm (LWHH, 56 ppm), whereas a considerable fraction remains surface oriented (Fig. S3 B).

When the peptide is reconstituted into DMPC at pH 5, two well-defined resonances at ∼80 and 195 ppm indicate that in-plane and transmembrane alignments co-exist, with the former being favored (Fig. S3, C and E). At pH 7.4, broad distributions are observed where ∼60–76% of the intensities are associated with resonances larger than the isotropic value of 120 ppm (Fig. S3, D and F). Notably, despite the broad distribution of the ¹⁵N resonances, the ³¹P NMR spectra of the DMPC lipids within the same samples are well oriented (Fig. S3, G and H).

Discussion

The ³¹P and ²H solid-state NMR spectra presented here indicate that the LAH4-L1 efficiently interacts with POPC/POPS 3:1 and DMPC lipid bilayers where, depending on the details of the experimental conditions, it can result in macroscopic phase changes. Interestingly, the pH- and P/L-ratio-dependent modifications of DMPC and POPC/POPS 3:1 closely resemble each other, with some subtle differences. The most pronounced phase transitions occur at pH 5, where addition of LAH4-L1 at ratios of 1:12.5 (DMPC at 37°C) or 1:25 (POPC/POPS 3:1 at 30°C) results in a large majority of phospholipids being oriented with their long axis perpendicular to the magnetic field direction (Figs. 1 H and 2 M). Further increasing the amount of peptide results in highly oriented ³¹P NMR spectra (Figs. 1 I and 2 N) where the absence of spectral intensities >0 ppm suggests a profound change in membrane properties (cf. below). At the highest peptide concentrations, isotropic resonances are observed (Figs. 1 K and 2 O). The tendency for isotropic resonances is considerably increased for gel-state DMPC membranes (Fig. 2), whereas it is absent when liquid crystalline bilayers are investigated at pH 7.4 (Figs. 1, A–D, and 2, A–F). Notably, at pH 7.4 and the highest peptide concentrations investigated here, partial alignment of the lipids with their long axes parallel to the magnetic field direction is observed (Figs. 1 E and 2 G).

At the same time, the ³¹P chemical shift anisotropy of the DMPC membranes decreases in a continuous manner with the addition of peptide (Fig. 3). In contrast, the chemical shift anisotropy of the POPC/POPS membranes, as measured by apparent δ⊥ position, exhibits a small increase until isotropic line shapes are obtained (Fig. 3). The chemical shift anisotropy reflects changes in the dynamic properties of the phospholipid and/or their supramolecular assembly, including formation of micelles or isotropic bicelles (25, 26), but also conformational changes of the headgroup. Indeed, large conformational alterations of the POPC headgroup have been observed upon addition of cationic amphiphiles concomitant with an increase in chemical shielding anisotropies (48).

Notably, the ²H solid-state NMR spectra of the fatty acyl chains (Figs. 4 and S2) closely reflect the spectral changes of the ³¹P solid-state NMR spectra from the phospholipid headgroups (Figs. 1 and 2), indicating that changes in the supramolecular features of the membranes, rather than local conformational properties, contribute to a large extent to the spectral alterations. Taken together at pH 5 in the presence of LAH4-L1 at P/L ratios of ≥12.5 and <1:5 the magnetic alignment of phospholipids and the concomitant NMR spectral line shapes (Figs. 1 I and 2, M and N) closely resemble those observed when anisotropic bicelles form in the presence of detergents, short-chain phospholipids, or other amphipathic peptides (27, 29). The formation of bicelles that align in the magnetic field of the NMR spectrometer is a function not only of composition and hydration but also of temperature (49), and this has also been observed for the sample shown in Fig. 1 I. Within lipid bilayers, the fatty acyl chains exhibit a tendency to align parallel to each other, which results in the constructive summation of their diamagnetic susceptibility anisotropy (50). The resulting magnetic interaction tends to align the lipid long axes perpendicular to the field of the NMR spectrometer to minimize the potential magnetic energy (51), concomitant with a notable prolate deformation of spherical liposomes made from pure lipids (Fig. 2 H; (45)) or the magnetic alignment of anisotropic bicelles. On the other hand, the many transmembrane helices of bacteriorhodopsin result in the alignment of the purple membrane normal parallel to the magnetic field direction (52, 53). The oblate deformation of the vesicles at high P/L ratios observed at pH 7.4 (Figs. 1 E and 2 G) thereby correlates with the transmembrane helical insertion of LAH4-L1 (Fig. 6, A and B).

At room temperature, only at the acidic condition, an isotropic line shape of the phospholipid ³¹P solid-state NMR spectra is observed at P/L molar ratios of 1:5 (Figs. 1 K and 2 O). Similar spectral features have been associated with nanodiscs and isotropic bicelles (54), suggesting that with the successive addition of LAH4-L1, first anistropic bicellar structures form (Figs. 1 I and 2, M and N) that then become small enough to tumble fast in all directions when compared to the ³¹P chemical shift anisotropy (Figs. 1 K and 2 O).

The NMR spectra of gel-phase DMPC more easily show isotropic peak intensities in the presence of LAH4-L1, which matches well with significant contributions in the <200 nm range (55) when the hydrodynamic radii are measured by DLS (Fig. S1). Notably, the DLS data were recorded at ambient temperature, i.e., around the gel-to-liquid-crystalline phase transition temperature (23°C). Both NMR and DLS show that with the increased addition of LAH4-L1, the structures diminish in size to reach a 10 nm hydrodynamic radius at P/L ratios of 1:5 at both pH values and isotropic NMR intensities under all conditions investigated. Furthermore, both approaches indicate that at pH 7.4, the supramolecular LAH4-L1/DMPC assemblies have a higher hydrodynamic radius when compared to pH 5.

The LAH4-L1-induced phase transitions of membranes can be explained by the pH-dependent physico-chemical properties of the peptide carrying four histidines. In dodecylphosphocholine micellar environments, the four histidines of LAH4-L1 exhibit pK values of 5.7, 5.8, 6.2, and 6.2 (Fig. S4). Whereas the polar histidines confer an amphipathic character to the helical structure at both pH values (11), its hydrophobic moment significantly increases when the histidine side chains turn cationic at low pH (20, 21). At the same time, the peptide increases its charge from nominally +5 to about +9 (8). As a consequence, less peptide is needed to compensate for the negative charges of the cargo (such as nucleic acids), and a large portion of LAH4 peptides is released from these polyanions (23). Concomitant with these physico-chemical changes, the peptide helix preferentially aligns in a transmembrane fashion at neutral conditions but adopts an orientation parallel to the membrane surface at low pH (Fig. 6, A–D), similar to the topological changes observed for LAH4 (1, 12, 56). Interestingly, whereas this transition is gradual in DMPC and POPC (Fig. S3), it is complete in the presence of POPC/POPS 3:1, where virtually all ¹⁵N chemical shift intensity appears in the transmembrane region at neutral pH and all in-planar intensity at pH 5 (Fig. 6, A–D). Similarly, the buffer, the presence of phosphatidylglycerol lipids, and the fatty acyl chain composition have been shown to have an influence on the topology of LAH4 (12) and of the antimicrobial peptide PGLa (57, 58), indicating that small energetic contributions can shift the topological equilibria of some amphipathic peptides (59). Although currently we cannot tell which type of interaction is responsible for the subtle differences in the alignment distribution of LAH4 peptides in POPC/POPG (12), POPC/POPS, and pure phosphatidylcholine membranes, previous investigations suggest that POPS interacts with LAH4-L1 in a manner that is different from the membrane partitioning of other peptides and different from its interactions with pure POPC (37).

Previously, mixtures of phospholipids and amphipathic peptides have been shown to have the potential to form bicellar phases that can orient in the magnetic field. Thus the antimicrobial peptide magainin 2 results in oriented phospholipid membranes at P/L ratios that are similar to those observed in this study (29). Furthermore, mixtures of phosphatidylcholines with amphipathic sequences derived from apolipoprotein A-1 form disk-like structures that, depending on the P/L ratio, exhibit either magnetic alignment (E. Salnikov and B.B., unpublished data) or isotropic NMR spectra (54). The naturally occurring apolipoprotein A, an important part of low-density lipoproteins, is the origin of the membrane scaffolding protein used to create supramolecular nanodiscs of well-defined dimension (60). These have become a popular tool for solution-state NMR investigations of membrane proteins, and polypeptide-lipid mixtures have stimulated the use of amphipathic styrene-maleic acid polymers to form nanodisc-like structures for NMR and other applications (61).

For the transfection experiments, non-covalent complexes are formed at pH 7.4 which, depending on the detailed conditions during their preparation outside the cells, are 100 nm to several micrometers in diameter and exhibit an overall positive surface potential (22). The supramolecular structures thus prepared are composed of ∼1 peptide/2 basepairs (23). These peptide/DNA transfection complexes enter the cells via an endosomal pathway (21), where acidification causes the histidine side chains to become cationic, yields a more relaxed packing of the remaining complexes (62), and results in the release of almost half of the LAH4 peptides from the DNA (23). It has been estimated that the amount of liberated peptide exceeds the number of endosomal lipids such that the high P/L ratios used here can in principle be obtained (23).

In this article, we demonstrated that LAH4-L1 exhibits membrane-disruptive properties at acidic pH, when the LAH4-L1 helices orient parallel to the membrane surface (Fig. 6, C and D) and/or when the membranes are in the gel state (Figs. 1 and 2). The mixed POPC/POPS 3:1 lipid bilayer was chosen as a well-defined model membrane representing the composition of zwitterionic and anionic lipids of the endosomal membrane (63). At acidic conditions, LAH4-L1 adopts in-planar configurations where the peptides pack the headgroup region densely with lipid but do not fill the hydrophobic interior of the membranes. Thus, they interact with the membrane like other inverted cone-shaped molecules (e.g., detergents). These are known to cause a decreased order parameter of the phospholipid fatty acyl chains, membrane thinning, and considerable curvature strain on the membrane (30, 64), which lead to membrane disordering (Figs. 5, B and D and 6 H) and ultimately phase transitions into different supramolecular arrangements (Figs. 1, 2, 4, and S2). The decrease in order parameter is most pronounced for POPS at pH 5 (Fig. 5 B), when the peptide is in-planar (Fig. 6, C and D), and considerably less pronounced at pH 7.4 (Fig. 5, A and B), when the peptide is fully transmembrane (Fig. 6, A and B). The effect is more pronounced for POPS than DMPC, which may be due to the selective interaction of LAH4 with this anionic lipid (4, 37), as well as association into mesophase arrangements, as has been observed for LAH4 (19). Therefore, the local peptide density around POPS may be much higher than expected from the nominal P/L ratio. Furthermore, differences in the average peptide topology are observed when the two membranes are compared to each other, with a higher proportion of in-plane oriented peptide in the POPC/POPS membrane at pH 5 (cf. Figs. 6 D and S3, C and E).

Interestingly, at pH 7.4, the POPS order parameter decreases in a concentration-dependent manner upon addition of peptide before the values close to the reference are observed at a P/L ratio of 1:12.5 (Fig. 5 A). This observation is suggestive of a structural rearrangement of the supramolecular complex, such as peptide oligomerization or formation of a bicellar rim structure.

Taken together, at low pH, the peptides exhibit detergent-like properties (65) that at the high P/L ratios lead to membrane lysis and the formation of bicelle-, nanodisc-, and/or micelle- like structures (Figs. 1, 2, 4 and S2). In contrast, at neutral pH, their toxicity for cells is much reduced as they are rather inefficient in disrupting membranes (Figs. 1, A–E, and 2, A–G), because they remain associated with their cargo, e.g., within the DNA transfection complexes (23), or they tend to form oligomers or other forms of aggregates (18). Thereby, LAH4-L1 has been used for the highly efficient transport and intracellular release of the cargo, such as nucleic acids (5, 9, 21), polypeptides (5, 8), viruses (2, 7), and quantum dots (6), into eukaryotic cell lines. Whereas endosomal release is a step that has been found to severely limit the cell-penetrating capabilities of other peptides (24), the non-covalent nature of peptide association with the complex and the balanced composition of the LAH4-L1 peptide, with amino acid side chains changing their protonation state in the endosome (His) and others that remain unaffected (Lys), are key features during the process (8). Upon release from the endosome, siRNA, proteins, and peptides have already reached their cytoplasmic destination, whereas other biological macromolecules, such as DNA, still have to continue their journey and enter the nucleus.

Author Contributions

J.W. and N.V. performed experiments. P.B. and J.R. performed NMR experiments and helped with technical issues. C.A. and N.H. analyzed experiments in quantitative detail. R.S. and B.B. designed experiments. J.W. and B.B. wrote the article.

Editor: Kalina Hristova.

Supporting Citations

Reference (66) appears in the Supporting Material.