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The pH-low insertion peptide (pHLIP) is an anionic membrane-active peptide with promising potential for applications in imaging of cancer tumors and targeted delivery of chemotherapeutics. The key advantage of pHLIP lies in its acid sensitivity: in acidic cellular environments, pHLIP can insert unidirectionally into the plasma membrane. Partitioning–folding coupling is triggered by titration of the acidic residues in pHLIP, transforming pHLIP from a hydrophilic to a hydrophobic peptide. Despite this knowledge, the reverse pathway that leads to exit of the peptide from the plasma membrane is poorly understood. Our hypothesis is that sequential deprotonation of pHLIP is a prerequisite for exit of the peptide from the plasma membrane. We carried out molecular dynamics (MD) simulations to characterize the effect that deprotonation of the acidic residues of pHLIP has on the stability of the peptide when inserted into a model lipid bilayer of 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC). Initiation of the exit mechanism is facilitated by a complex relationship between the peptide, bulk solvent, and the membrane environment. As the N-terminal acidic residues of pHLIP are deprotonated, localized loss of helicity drives unfolding of the peptide and more pronounced interactions with the bilayer at the lipid–water interface. Deprotonation of the C-terminal acidic residues (D25, D31, D33, and E34) leads to further loss of secondary structure distal from the C-terminus, as well as formation of a water channel that stabilizes the orientation of pHLIP parallel to the membrane normal. Together, these results help explain how stabilization of intermediates between the surface-bound and inserted states of pHLIP occur and provide insights into rational design of pHLIP variants with modified abilities of insertion.
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This content will become publicly available on July 1, 2026
Insights into Membrane Damage by α-Helical and β-Sheet Peptides
Peptide-induced disruption of lipid membranes is central to both amyloid diseases and the activity of antimicrobial peptides. Here, we combine all-atom molecular dynamics simulations with biophysical experiments to investigate how four amphipathic peptides interact with lipid bilayers. All peptides adsorb on the membrane surface. Peptide M01 [Ac-(FKFE)2-NH2] self-assembles into β-sheet nanofibrils that span both leaflets of the membrane, creating water-permeable channels. The other three peptides adopt α-helical structures at the water–lipid interface. Peptide M02 [Ac-FFKKFFEE-NH2], a sequence isomer of M01, does not form β-sheet aggregates and is too short to span the bilayer, resulting in no observable water permeation across the membrane. Peptides M03 and M04 are α-helical isomers long enough to span the bilayer, with a polar face that allows the penetration of water deep inside the membrane. For the M03 peptide [Ac-(FFKKFFEE)2-NH2], insertion into the bilayer starts with the nonpolar N-terminal amino acids penetrating the hydrophobic core of the bilayer, while electrostatic interactions hold negative residues at the C-terminus on the membrane surface. The M04 peptide, [Ac-FFKKFFEEFKKFFEEF-NH2], is made by relocating a single nonpolar residue from the central region of M03 to the C-terminus. This nonpolar residue becomes unfavorably exposed to the solvent upon insertion of the N-terminal region of the peptide into the membrane. Consequently, higher concentrations of M04 peptides are required to induce water permeation compared to M03. Overall, our comparative analysis reveals how subtle rearrangements of polar and nonpolar residues modulate peptide-induced water permeation. This provides mechanistic insights relevant to amyloid pathology and antimicrobial peptide design.
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- PAR ID:
- 10621608
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Biomolecules
- Volume:
- 15
- Issue:
- 7
- ISSN:
- 2218-273X
- Page Range / eLocation ID:
- 973
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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