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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 May 1, 2026
Biophysics of pH-Driven Membrane Insertion: A Review of the pHLIP Peptide
The pH Low Insertion Peptide (pHLIP) is a useful model for exploring the biophysical chemistry of pH-driven membrane insertion and folding. This review discusses recent advancements in understanding the molecular mechanisms underlying pHLIP behavior, focusing on its ability to transition from a soluble, unstructured state to a membrane-inserted α-helix. Protonation of acidic residues, changes in peptide hydrophobicity, and interactions with the lipid bilayer, are described. Recent studies using NMR, infrared spectroscopy, and molecular dynamics simulations have provided a stepwise mechanistic model of the coupled folding and insertion process including its intermediate states present under different pH conditions. In addition, pHLIP ability to selectively target acidic microenvironments, such as those found in tumors, has made it a promising tool for biomedical applications. We provide an overview of recent fundamental studies and applications and discuss how future work can benefit from combining advanced experimental and computational approaches to refine our understanding of the peptide’s structure–function relationships.
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- Award ID(s):
- 2129209
- PAR ID:
- 10645459
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- The Journal of Physical Chemistry B
- Volume:
- 129
- Issue:
- 17
- ISSN:
- 1520-6106
- Page Range / eLocation ID:
- 4123 to 4132
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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