Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides

Gregory Wiedman, William C. Wimley, Kalina Hristova

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


In this work, we sought to rationally design membrane-active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane-active peptides, pHlip and GALA. We created two hybrid sequences, MelP5-Δ4 and MelP5-Δ6, by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macromolecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH sensitivity into its sequence. We conclude that the properties of pHlip, GALA, and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane-active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field.

Original languageEnglish (US)
Pages (from-to)951-957
Number of pages7
JournalBiochimica et Biophysica Acta - Biomembranes
Issue number4
StatePublished - Apr 2015


  • Membrane
  • Peptides
  • pH sensitivity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology


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