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 N 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,P and H 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/H-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.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers

“…The interfacial alignment also agrees with the amphipathic character of the peptide sequence . In‐planar helix alignments have previously also been observed for a multitude of charged and uncharged amphipathic sequences …”

“…When the 31 P NMR spectra of the three samples were investigated, a predominant intensity was observed at 28 ppm with much smaller intensities reaching up to −16 ppm (Figure B, D, F), thus indicating that the phospholipid is in a liquid crystalline state, and the bilayers are well oriented . Interestingly, in contrast to the large increase in signal intensities <30 ppm that occur when the peptide‐lipid‐ratio of amphipathic cationic antimicrobial sequences is increased >1:50, in the presence of trichogin, closely related 31 P NMR line shapes are observed for all three peptide‐to‐lipid ratios (Figure B, D, F). Overall, even at the high peptide concentrations, the peptide and lipids aligned well, thereby resembling the behavior of alamethicin .…”

The Mechanisms of Action of Cationic Antimicrobial Peptides Refined by Novel Concepts from Biophysical Investigations

“…The most cited is the formation of toroidal pores (Ludtke et al 1996;Matsuzaki 1998), a dense "carpet" of peptides covering the membrane surface that causes lysis (Shai 1999), or small aggregates without specific structure within the membrane (Jenssen et al 2006). Whereas on the one hand, occasionally channel-like events are recorded in electrophysiological experiment (Christensen et al 1988;Duclohier et al 1989;Cruciani et al 1991), at high peptide concentrations, worm-like structures, disk-shaped particles, or micelles have been observed (Hallock et al 2002;Bechinger and Lohner 2006;Wolf et al 2017). This wide variety of observations can be taken into consideration by the differential shape of lipids (Fig.…”

Supramolecular Organization of Apolipoprotein-A-I-Derived Peptides within Disc-like Arrangements

“…Because the size of the MSP nanodisc is a sole function of the number and length of the helical segments that are interconnected by proline and glycine residues (24), the MSP can be considered to consist of a string of independent helices without a complex tertiary fold. Whereas the geometry of the nanodisc is defined through the covalent linkages between the helices composing the apolipoprotein, amphipathic helices from this sequence or from a different origin can also be mixed with lipids, where the size of the nanodisc structures can be tuned by changing the peptideto-lipid ratio (28). The amphipathic peptides thereby take the role of detergents or short-chain lipids in classical bicellar complexes (19).…”